Mendel: From genes to genome.

Two hundred years after the birth of Gregor Mendel, it is an appropriate time to reflect on recent developments in the discipline of genetics, particularly advances relating to the prescient friar's model species, the garden pea (Pisum sativum L.). Mendel's study of seven characteristics established the laws of segregation and independent assortment. The genes underlying four of Mendel's loci (A, LE, I, and R) have been characterized at the molecular level for over a decade. However, the three remaining genes, influencing pod color (GP), pod form (V/P), and the position of flowers (FA/FAS), have remained elusive for a variety of reasons, including a lack of detail regarding the loci with which Mendel worked. Here, we discuss potential candidate genes for these characteristics, in light of recent advances in the genetic resources for pea. These advances, including the pea genome sequence and reverse-genetics techniques, have revitalized pea as an excellent model species for physiological-genetic studies. We also discuss the issues that have been raised with Mendel's results, such as the recent controversy regarding the discrete nature of the characters that Mendel chose and the perceived overly-good fit of his segregations to his hypotheses. We also consider the relevance of these controversies to his lasting contribution. Finally, we discuss the use of Mendel's classical results to teach and enthuse future generations of geneticists, not only regarding the core principles of the discipline, but also its history and the role of hypothesis testing.

Trehalose 6-phosphate promotes seed filling by activating auxin biosynthesis.

Plants undergo several developmental transitions during their life cycle. One of these, the differentiation of the young embryo from a meristem-like structure into a highly specialized storage organ, is believed to be controlled by local connections between sugars and hormonal response systems. However, we know little about the regulatory networks underpinning the sugar-hormone interactions in developing seeds. By modulating the trehalose 6-phosphate (T6P) content in growing embryos of garden pea (Pisum sativum), we investigate here the role of this signaling sugar during the seed-filling process. Seeds deficient in T6P are compromised in size and starch production, resembling the wrinkled seeds studied by Gregor Mendel. We show also that T6P exerts these effects by stimulating the biosynthesis of the pivotal plant hormone, auxin. We found that T6P promotes the expression of the auxin biosynthesis gene TRYPTOPHAN AMINOTRANSFERASE RELATED2 (TAR2), and the resulting effect on auxin concentrations is required to mediate the T6P-induced activation of storage processes. Our results suggest that auxin acts downstream of T6P to facilitate seed filling, thereby providing a salient example of how a metabolic signal governs the hormonal control of an integral phase transition in a crop plant.

Linking Auxin with Photosynthetic Rate via Leaf Venation.

Land plants lose vast quantities of water to the atmosphere during photosynthetic gas exchange. In angiosperms, a complex network of veins irrigates the leaf, and it is widely held that the density and placement of these veins determines maximum leaf hydraulic capacity and thus maximum photosynthetic rate. This theory is largely based on interspecific comparisons and has never been tested using vein mutants to examine the specific impact of leaf vein morphology on plant water relations. Here we characterize mutants at the Crispoid (Crd) locus in pea (Pisum sativum), which have altered auxin homeostasis and activity in developing leaves, as well as reduced leaf vein density and aberrant placement of free-ending veinlets. This altered vein phenotype in crd mutant plants results in a significant reduction in leaf hydraulic conductance and leaf gas exchange. We find Crispoid to be a member of the YUCCA family of auxin biosynthetic genes. Our results link auxin biosynthesis with maximum photosynthetic rate through leaf venation and substantiate the theory that an increase in the density of leaf veins coupled with their efficient placement can drive increases in leaf photosynthetic capacity.

Evidence that auxin is required for normal seed size and starch synthesis in pea.

In recent years the biosynthesis of auxin has been clarified with the aid of mutations in auxin biosynthesis genes. However, we know little about the effects of these mutations on the seed-filling stage of seed development. Here we investigate a key auxin biosynthesis mutation of the garden pea, which results in auxin deficiency in developing seeds. We exploit the large seed size of this model species, which facilitates the measurement of compounds in individual seeds. The mutation results in small seeds with reduced starch content and a wrinkled phenotype at the dry stage. The phenotypic effects of the mutation were fully reversed by introduction of the wild-type gene as a transgene, and partially reversed by auxin application. The results indicate that auxin is required for normal seed size and starch accumulation in pea, an important grain legume crop.

Auxin Biosynthesis: Are the Indole-3-Acetic Acid and Phenylacetic Acid Biosynthesis Pathways Mirror Images?

The biosynthesis of the main auxin in plants (indole-3-acetic acid [IAA]) has been elucidated recently and is thought to involve the sequential conversion of Trp to indole-3-pyruvic acid to IAA However, the pathway leading to a less well studied auxin, phenylacetic acid (PAA), remains unclear. Here, we present evidence from metabolism experiments that PAA is synthesized from the amino acid Phe, via phenylpyruvate. In pea (Pisum sativum), the reverse reaction, phenylpyruvate to Phe, is also demonstrated. However, despite similarities between the pathways leading to IAA and PAA, evidence from mutants in pea and maize (Zea mays) indicate that IAA biosynthetic enzymes are not the main enzymes for PAA biosynthesis. Instead, we identified a putative aromatic aminotransferase (PsArAT) from pea that may function in the PAA synthesis pathway.

EARLY FLOWERING3 Regulates Flowering in Spring Barley by Mediating Gibberellin Production and FLOWERING LOCUS T Expression.

EARLY FLOWERING3 (ELF3) is a circadian clock gene that contributes to photoperiod-dependent flowering in plants, with loss-of-function mutants in barley (Hordeum vulgare), legumes, and Arabidopsis thaliana flowering early under noninductive short-day (SD) photoperiods. The barley elf3 mutant displays increased expression of FLOWERING LOCUS T1 (FT1); however, it remains unclear whether this is the only factor responsible for the early flowering phenotype. We show that the early flowering and vegetative growth phenotypes of the barley elf3 mutant are strongly dependent on gibberellin (GA) biosynthesis. Expression of the central GA biosynthesis gene, GA20oxidase2, and production of the bioactive GA, GA1, were significantly increased in elf3 leaves under SDs, relative to the wild type. Inhibition of GA biosynthesis suppressed the early flowering of elf3 under SDs independently of FT1 and was associated with altered expression of floral identity genes at the developing apex. GA is also required for normal flowering of spring barley under inductive photoperiods, with chemical and genetic attenuation of the GA biosynthesis and signaling pathways suppressing inflorescence development under long-day conditions. These findings illustrate that GA is an important floral promoting signal in barley and that ELF3 suppresses flowering under noninductive photoperiods by blocking GA production and FT1 expression.

Sugar demand, not auxin, is the initial regulator of apical dominance.

For almost a century the plant hormone auxin has been central to theories on apical dominance, whereby the growing shoot tip suppresses the growth of the axillary buds below. According to the classic model, the auxin indole-3-acetic acid is produced in the shoot tip and transported down the stem, where it inhibits bud growth. We report here that the initiation of bud growth after shoot tip loss cannot be dependent on apical auxin supply because we observe bud release up to 24 h before changes in auxin content in the adjacent stem. After the loss of the shoot tip, sugars are rapidly redistributed over large distances and accumulate in axillary buds within a timeframe that correlates with bud release. Moreover, artificially increasing sucrose levels in plants represses the expression of BRANCHED1 (BRC1), the key transcriptional regulator responsible for maintaining bud dormancy, and results in rapid bud release. An enhancement in sugar supply is both necessary and sufficient for suppressed buds to be released from apical dominance. Our data support a theory of apical dominance whereby the shoot tip's strong demand for sugars inhibits axillary bud outgrowth by limiting the amount of sugar translocated to those buds.

Evidence That Chlorinated Auxin Is Restricted to the Fabaceae But Not to the Fabeae.

Auxin is a pivotal plant hormone, usually occurring in the form of indole-3-acetic acid (IAA). However, in maturing pea (Pisum sativum) seeds, the level of the chlorinated auxin, 4-chloroindole-3-acetic acid (4-Cl-IAA), greatly exceeds that of IAA. A key issue is how plants produce halogenated compounds such as 4-Cl-IAA. To better understand this topic, we investigated the distribution of the chlorinated auxin. We show for the first time, to our knowledge, that 4-Cl-IAA is found in the seeds of Medicago truncatula, Melilotus indicus, and three species of Trifolium. Furthermore, we found no evidence that Pinus spp. synthesize 4-Cl-IAA in seeds, contrary to a previous report. The evidence indicates a single evolutionary origin of 4-Cl-IAA synthesis in the Fabaceae, which may provide an ideal model system to further investigate the action and activity of halogenating enzymes in plants.

Shoot-derived abscisic acid promotes root growth.

The phytohormone abscisic acid (ABA) plays a major role in regulating root growth. Most work to date has investigated the influence of root-sourced ABA on root growth during water stress. Here, we tested whether foliage-derived ABA could be transported to the roots, and whether this foliage-derived ABA had an influence on root growth under well-watered conditions. Using both application studies of deuterium-labelled ABA and reciprocal grafting between wild-type and ABA-biosynthetic mutant plants, we show that both ABA levels in the roots and root growth in representative angiosperms are controlled by ABA synthesized in the leaves rather than sourced from the roots. Foliage-derived ABA was found to promote root growth relative to shoot growth but to inhibit the development of lateral roots. Increased root auxin (IAA) levels in plants with ABA-deficient scions suggest that foliage-derived ABA inhibits root growth through the root growth-inhibitor IAA. These results highlight the physiological and morphological importance, beyond the control of stomata, of foliage-derived ABA. The use of foliar ABA as a signal for root growth has important implications for regulating root to shoot growth under normal conditions and suggests that leaf rather than root hydration is the main signal for regulating plant responses to moisture.

Strigolactones stimulate internode elongation independently of gibberellins.

Strigolactone (SL) mutants in diverse species show reduced stature in addition to their extensive branching. Here, we show that this dwarfism in pea (Pisum sativum) is not attributable to the strong branching of the mutants. The continuous supply of the synthetic SL GR24 via the root system using hydroponics can restore internode length of the SL-deficient rms1 mutant but not of the SL-response rms4 mutant, indicating that SLs stimulate internode elongation via RMS4. Cytological analysis of internode epidermal cells indicates that SLs control cell number but not cell length, suggesting that SL may affect stem elongation by stimulating cell division. Consequently, SLs can repress (in axillary buds) or promote (in the stem) cell division in a tissue-dependent manner. Because gibberellins (GAs) increase internode length by affecting both cell division and cell length, we tested if SLs stimulate internode elongation by affecting GA metabolism or signaling. Genetic analyses using SL-deficient and GA-deficient or DELLA-deficient double mutants, together with molecular and physiological approaches, suggest that SLs act independently from GAs to stimulate internode elongation.

Septic Arthritis of the Spinal Facet Joint: Review of 117 Cases.

Background: Septic arthritis of the spinal facet joints is increasingly recognized in the era of magnetic resonance imaging, but its epidemiology, clinical features, management, and prognosis are ill-defined. Methods: We review 101 previously published cases and report 16 cases occurring at our institutions between 2006 and 2018. Results: Most patients presented with fever (60%) and back or neck pain (86%). Radiation into the hip, buttock, or limb was present in 34%. The lumbosacral vertebral segments were involved in 78% of cases. Most cases (64%) were due to Staphylococcus aureus. Bacteremia was present in 66% and paraspinal muscle abscesses in 54%. While epidural abscesses were present in 56%, neurologic complications were seen in only 9%, likely because most abscesses arose below the conus medullaris. Neurologic complications were more common with cervical or thoracic involvement than lumbosacral (32% vs 2%, P < .0001). Extraspinal infection, such as endocarditis, was identified in only 22% of cases. An overall 98% of patients survived, with only 5% having neurologic sequelae. Conclusions: Septic arthritis of the facet joint is a distinct clinical syndrome typically involving the lumbar spine and is frequently associated with bacteremia, posterior epidural abscesses, and paraspinal pyomyositis. Neurologic outcomes are usually good with medical management alone.

Fate of perfluoroalkyl and polyfluoroalkyl substances (PFAS) through two full-scale wastewater sludge incinerators.

Perfluoroalkyl and polyfluoroalkyl substances (PFAS) are an emerging issue in wastewater treatment. High-temperature thermal processes, incineration being time-tested, offer the opportunity to destroy and change the composition of PFAS. The fate of PFAS has been documented through wastewater sludge incinerators, including a multiple hearth furnace (MHF) and a fluidized bed furnace (FBF). The dewatered wastewater sludge feedstock averaged 247- and 1280-µmol targeted PFAS per sample run in MHF and FBF feed, respectively. Stack emissions (reportable for all targeted PFAS from MHF only) averaged 5% of that value with shorter alkyl chain compounds comprising the majority of the targeted PFAS. Wet scrubber water streams accumulated nonpolar fluorinated organics from the furnace exhaust with an average of 0.740- and 0.114-mol F- per sample run, for the MHF and FBF, respectively. Simple alkane PFAS measured at the stack represented 0.5%-4.5% of the total estimated facility greenhouse gas emissions. PRACTITIONER POINTS: The MHF emitted six short chain PFAS from the stack, which were shorter alkyl chain compounds compared with sludge PFAS. The FBF did not consistently emit reportable PFAS from the stack, but contamination complicated the assessment. Five percent of the MHF sludge molar PFAS load was reported in the stack. MHF and FBF wet scrubber water streams accumulated nonpolar fluorinated organics from the furnace exhaust. Ultra-short volatile alkane PFAS measured at the stack represented 0.5%-4.5% of the estimated facility greenhouse gas emissions.

Impaired auxin biosynthesis in the defective endosperm18 mutant is due to mutational loss of expression in the ZmYuc1 gene encoding endosperm-specific YUCCA1 protein in maize.

The phytohormone auxin (indole-3-acetic acid [IAA]) plays a fundamental role in vegetative and reproductive plant development. Here, we characterized a seed-specific viable maize (Zea mays) mutant, defective endosperm18 (de18) that is impaired in IAA biosynthesis. de18 endosperm showed large reductions of free IAA levels and is known to have approximately 40% less dry mass, compared with De18. Cellular analyses showed lower total cell number, smaller cell volume, and reduced level of endoreduplication in the mutant endosperm. Gene expression analyses of seed-specific tryptophan-dependent IAA pathway genes, maize Yucca1 (ZmYuc1), and two tryptophan-aminotransferase co-orthologs were performed to understand the molecular basis of the IAA deficiency in the mutant. Temporally, all three genes showed high expression coincident with high IAA levels; however, only ZmYuc1 correlated with the reduced IAA levels in the mutant throughout endosperm development. Furthermore, sequence analyses of ZmYuc1 complementary DNA and genomic clones revealed many changes specific to the mutant, including a 2-bp insertion that generated a premature stop codon and a truncated YUC1 protein of 212 amino acids, compared with the 400 amino acids in the De18. The putative, approximately 1.5-kb, Yuc1 promoter region also showed many rearrangements, including a 151-bp deletion in the mutant. Our concurrent high-density mapping and annotation studies of chromosome 10, contig 395, showed that the De18 locus was tightly linked to the gene ZmYuc1. Collectively, the data suggest that the molecular changes in the ZmYuc1 gene encoding the YUC1 protein are the causal basis of impairment in a critical step in IAA biosynthesis, essential for normal endosperm development in maize.

Biosynthesis of the halogenated auxin, 4-chloroindole-3-acetic acid.

Seeds of several agriculturally important legumes are rich sources of the only halogenated plant hormone, 4-chloroindole-3-acetic acid. However, the biosynthesis of this auxin is poorly understood. Here, we show that in pea (Pisum sativum) seeds, 4-chloroindole-3-acetic acid is synthesized via the novel intermediate 4-chloroindole-3-pyruvic acid, which is produced from 4-chlorotryptophan by two aminotransferases, TRYPTOPHAN AMINOTRANSFERASE RELATED1 and TRYPTOPHAN AMINOTRANSFERASE RELATED2. We characterize a tar2 mutant, obtained by Targeting Induced Local Lesions in Genomes, the seeds of which contain dramatically reduced 4-chloroindole-3-acetic acid levels as they mature. We also show that the widespread auxin, indole-3-acetic acid, is synthesized by a parallel pathway in pea.

Plant hormones in arbuscular mycorrhizal symbioses: an emerging role for gibberellins.

BACKGROUND AND AIMS: Arbuscular mycorrhizal symbioses are important for nutrient acquisition in >80 % of terrestrial plants. Recently there have been major breakthroughs in understanding the signals that regulate colonization by the fungus, but the roles of the known plant hormones are still emerging. Here our understanding of the roles of abscisic acid, ethylene, auxin, strigolactones, salicylic acid and jasmonic acid is discussed, and the roles of gibberellins and brassinosteroids examined. METHODS: Pea mutants deficient in gibberellins, DELLA proteins and brassinosteroids are used to determine whether fungal colonization is altered by the level of these hormones or signalling compounds. Expression of genes activated during mycorrhizal colonization is also monitored. KEY RESULTS: Arbuscular mycorrhizal colonization of pea roots is substantially increased in gibberellin-deficient na-1 mutants compared with wild-type plants. This is reversed by application of GA3. Mutant la cry-s, which lacks gibberellin signalling DELLA proteins, shows reduced colonization. These changes were parallelled by changes in the expression of genes associated with mycorrhizal colonization. The brassinosteroid-deficient lkb mutant showed no change in colonization. CONCLUSIONS: Biologically active gibberellins suppress arbuscule formation in pea roots, and DELLA proteins are essential for this response, indicating that this role occurs within the root cells.

Blastomycosis in New England: 5 Cases and a Review.

The geographic range of blastomycosis is thought to include New England, but documentation is sparse. We report 5 cases of infection with Blastomyces dermatitidis that were likely acquired in New England between 2011 and 2021. Our experience suggests that chart coding for the diagnosis of blastomycosis is imprecise and that mandatory reporting might help resolve uncertainties about the prevalence and extent of blastomycosis.

Plant Development in the Garden Pea as Revealed by Mutations in the Crd/PsYUC1 Gene.

In common with other plant species, the garden pea (Pisum sativum) produces the auxin indole-3-acetic acid (IAA) from tryptophan via a single intermediate, indole-3-pyruvic acid (IPyA). IPyA is converted to IAA by PsYUC1, also known as Crispoid (Crd). Here, we extend our understanding of the developmental processes affected by the Crd gene by examining the phenotypic effects of crd gene mutations on leaves, flowers, and roots. We show that in pea, Crd/PsYUC1 is important for the initiation and identity of leaflets and tendrils, stamens, and lateral roots. We also report on aspects of auxin deactivation in pea.