Evidence for within-species transition between drought response strategies in Nicotiana benthamiana.

Nicotiana benthamiana is predominantly distributed in arid habitats across northern Australia. However, none of six geographically isolated accessions shows obvious xerophytic morphological features. To investigate how these tender-looking plants withstand drought, we examined their responses to water deprivation, assessed phenotypic, physiological, and cellular responses, and analysed cuticular wax composition and wax biosynthesis gene expression profiles. Results showed that the Central Australia (CA) accession, globally known as a research tool, has evolved a drought escape strategy with early vigour, short life cycle, and weak, water loss-limiting responses. By contrast, a northern Queensland (NQ) accession responded to drought by slowing growth, inhibiting flowering, increasing leaf cuticle thickness, and altering cuticular wax composition. Under water stress, NQ increased the heat stability and water impermeability of its cuticle by extending the carbon backbone of cuticular long-chain alkanes from c. 25 to 33. This correlated with rapid upregulation of at least five wax biosynthesis genes. In CA, the alkane chain lengths (c. 25) and gene expression profiles remained largely unaltered. This study highlights complex genetic and environmental control over cuticle composition and provides evidence for divergence into at least two fundamentally different drought response strategies within the N. benthamiana species in < 1 million years.

The evolving role of abscisic acid in cell function and plant development over geological time.

Abscisic acid (ABA) is found in a wide diversity of organisms, yet we know most about the hormonal action of this compound in the ecologically dominant and economically important angiosperms. In angiosperms, ABA regulates a suite of critical responses from desiccation tolerance through to seed dormancy and stomatal closure. Work exploring the function of key genes in the ABA signalling pathway of angiosperms has revealed that this signal transduction pathway is ancient, yet considerable change in the physiological roles of this hormone have occurred over geological time. With recent advances in our capacity to characterise gene function in non-angiosperms we are on the cusp of revealing the origins of this critical hormonal signalling pathway in plants, and understanding how a simple hormone may have shaped land plant diversity, ecology and adaptation over the past 500 million years.

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.

The genetic architecture of flowering time changes in pea from wild to crop.

Change in phenology has been an important component in crop evolution, and selection for earlier flowering through a reduction in environmental sensitivity has helped broaden adaptation in many species. Natural variation for flowering in domesticated pea (Pisum sativum L.) has been noted and studied for decades, but there has been no clear account of change relative to its wild progenitor. Here we examined the genetic control of differences in flowering time between wild P. sativum ssp. humile and a typical late-flowering photoperiodic P. s. sativum accession in a recombinant inbred population under long and short photoperiods. Our results confirm the importance of the major photoperiod sensitivity locus Hr/PsELF3a and identify two other loci on chromosomes 1 (DTF1) and 3 (DTF3) that contribute to earlier flowering in the domesticated line under both photoperiods. The domesticated allele at a fourth locus on chromosome 6 (DTF6) delays flowering under long days only. Map positions, inheritance patterns, and expression analyses in near-isogenic comparisons imply that DTF1, DTF3, and DTF6 represent gain-of-function alleles of the florigen/antiflorigen genes FTa3, FTa1, and TFL1c/LF, respectively. This echoes similar variation in chickpea and lentil, and suggests a conserved route to reduced photoperiod sensitivity and early phenology in temperate pulses.

From reproduction to production, stomata are the master regulators.

The best predictor of leaf level photosynthetic rate is the porosity of the leaf surface, as determined by the number and aperture of stomata on the leaf. This remarkable correlation between stomatal porosity (or diffusive conductance to water vapour gs ) and CO2 assimilation rate (A) applies to all major lineages of vascular plants (Figure 1) and is sufficiently predictable that it provides the basis for the model most widely used to predict water and CO2 fluxes from leaves and canopies. Yet the Ball-Berry formulation is only a phenomenological approximation that captures the emergent character of stomatal behaviour. Progressing to a more mechanistic prediction of plant gas exchange is challenging because of the diversity of biological components regulating stomatal action. These processes are the product of more than 400 million years of co-evolution between stomatal, vascular and photosynthetic tissues. Both molecular and structural components link the abiotic world of the whole plant with the turgor pressure of the epidermis and guard cells, which ultimately determine stomatal pore size and porosity to water and CO2 exchange (New Phytol., 168, 2005, 275). In this review we seek to simplify stomatal behaviour by using an evolutionary perspective to understand the principal selective pressures involved in stomatal evolution, thus identifying the primary regulators of stomatal aperture. We start by considering the adaptive process that has locked together the regulation of water and carbon fluxes in vascular plants, finally examining specific evidence for evolution in the proteins responsible for regulating guard cell turgor.

Continental-scale distribution and diversity of Ceratobasidium orchid mycorrhizal fungi in Australia.

BACKGROUND AND AIMS: Mycorrhizal fungi are a critical component of the ecological niche of most plants and can potentially constrain their geographical range. Unlike other types of mycorrhizal fungi, the distributions of orchid mycorrhizal fungi (OMF) at large spatial scales are not well understood. Here, we investigate the distribution and diversity of Ceratobasidium OMF in orchids and soils across the Australian continent. METHODS: We sampled 217 Ceratobasidium isolates from 111 orchid species across southern Australia and combined these with 311 Ceratobasidium sequences from GenBank. To estimate the taxonomic diversity of Ceratobasidium associating with orchids, phylogenetic analysis of the ITS sequence locus was undertaken. Sequence data from the continent-wide Australian Microbiome Initiative were used to determine the geographical range of operational taxonomic units (OTUs) detected in orchids, with the distribution and climatic correlates of the two most frequently detected OTUs modelled using MaxEnt. KEY RESULTS: We identified 23 Ceratobasidium OTUs associating with Australian orchids, primarily from the orchid genera Pterostylis, Prasophyllum, Rhizanthella and Sarcochilus. OTUs isolated from orchids were closely related to, but distinct from, known pathogenic fungi. Data from soils and orchids revealed that ten of these OTUs occur on both east and west sides of the continent, while 13 OTUs were recorded at three locations or fewer. MaxEnt models suggested that the distributions of two widespread OTUs are correlated with temperature and soil moisture of the wettest quarter and far exceeded the distributions of their host orchid species. CONCLUSIONS: Ceratobasidium OMF with cross-continental distributions are common in Australian soils and frequently have geographical ranges that exceed that of their host orchid species, suggesting these fungi are not limiting the distributions of their host orchids at large spatial scales. Most OTUs were distributed within southern Australia, although several OTUs had distributions extending into central and northern parts of the continent, illustrating their tolerance of an extraordinarily wide range of environmental conditions.

Independent genetic control of drought resistance, recovery, and growth of Eucalyptus globulus seedlings.

Drought is a major stress impacting forest ecosystems worldwide. We utilized quantitative trait loci (QTL) analysis to study the genetic basis of variation in (a) drought resistance and recovery and (b) candidate traits that may be associated with this variation in the forest tree Eucalyptus globulus. QTL analysis was performed using a large outcrossed F2 mapping population from which 300 trees were phenotyped based on the mean performance of their open-pollinated F3 progeny. Progenies were grown in a glasshouse in a randomized complete block design. A subset of seedlings was subjected to a drought treatment after which they were rewatered and scored for damage and growth postdrought. Nondroughted seedlings were assessed for growth traits as well as lignotuber size and resprouting following severe damage to the main stem. QTL were detected for most traits. Importantly, independent QTL were detected for (a) drought damage and plant size, (b) drought damage and growth recovery, and (c) lignotuber size and resprouting capacity. Such independence argues that trade-offs are unlikely to be a major limitation to the response to selection and at the early life history stage studied; there are opportunities to improve resilience to drought without adverse effects on productivity.

Specific mycorrhizal associations involving the same fungal taxa in common and threatened Caladenia (Orchidaceae): implications for conservation.

BACKGROUND AND AIMS: In orchid conservation, quantifying the specificity of mycorrhizal associations, and establishing which orchid species use the same fungal taxa, is important for sourcing suitable fungi for symbiotic propagation and selecting sites for conservation translocation. For Caladenia subgenus Calonema (Orchidaceae), which contains 58 threatened species, we ask the following questions. (1) How many taxa of Serendipita mycorrhizal fungi do threatened species of Caladenia associate with? (2) Do threatened Caladenia share orchid mycorrhizal fungi with common Caladenia? (3) How geographically widespread are mycorrhizal fungi associated with Caladenia? METHODS: Fungi were isolated from 127 Caladenia species followed by DNA sequencing of the internal transcibed spacer (ITS) sequence locus. We used a 4.1-6 % sequence divergence cut-off range to delimit Serendipita operational taxonomic units (OTUs). We conducted trials testing the ability of fungal isolates to support germination and plant growth. A total of 597 Serendipita isolates from Caladenia, collected from across the Australian continent, were used to estimate the geographic range of OTUs. KEY RESULTS: Across the genus, Caladenia associated with ten OTUs of Serendipita (Serendipitaceae) mycorrhizal fungi. Specificity was high, with 19 of the 23 threatened Caladenia species sampled in detail associating solely with OTU A, which supported plants from germination to adulthood. The majority of populations of Caladenia associated with one OTU per site. Fungal sharing was extensive, with 62 of the 79 Caladenia sampled in subgenus Calonema associating with OTU A. Most Serendipita OTUs were geographically widespread. CONCLUSIONS: Mycorrhizal fungi can be isolated from related common species to propagate threatened Caladenia. Because of high specificity of most Caladenia species, only small numbers of OTUs typically need to be considered for conservation translocation. When selecting translocation sites, the geographic range of the fungi is not a limiting factor, and using related Caladenia species to infer the presence of suitable fungal OTUs may be feasible.

On the origins of osmotically driven stomatal movements.

Contents Summary 84 I. Introduction 84 II. Stomatal form and biomechanics 85 III. Stomatal function 86 IV. Evolution of guard cell ion channels 87 V. Conclusions 88 Acknowledgements 88 Author contributions 88 References 88 SUMMARY: Stomatal pores with apertures that can be adjusted by changes in guard cell turgor have facilitated plant success in dry environments. We explore their evolutionary origins, considering recent findings from bryophytes. Unlike vascular plant stomata, which close to prevent water loss, bryophyte stomata become locked open to promote spore desiccation. We find that the families of ion channels, known to control stomatal movements in angiosperms, are ancient and represented across extant land plants. However, although angiosperm guard cells express specific ion channel genes, none appear specifically expressed in stomata-bearing moss tissues. Given the evolutionary shift in stomatal function from promotion to prevention of water loss, we postulate that ion channels adopted guard cell-specific functions after the divergence of bryophytes.

Identification of LATE BLOOMER2 as a CYCLING DOF FACTOR Homolog Reveals Conserved and Divergent Features of the Flowering Response to Photoperiod in Pea.

The molecular pathways responsible for the flowering response to photoperiod have been extensively studied in Arabidopsis thaliana and cereals but remain poorly understood in other major plant groups. Here, we describe a dominant mutant at the LATE BLOOMER2 (LATE2) locus in pea (Pisum sativum) that is late-flowering with a reduced response to photoperiod. LATE2 acts downstream of light signaling and the circadian clock to control expression of the main photoperiod-regulated FT gene, FTb2, implying that it plays a primary role in photoperiod measurement. Mapping identified the CYCLING DOF FACTOR gene CDFc1 as a strong candidate for LATE2, and the late2-1D mutant was found to carry a missense mutation in CDFc1 that impairs its capacity to bind to the blue-light photoreceptor FKF1 in yeast two-hybrid assays and delays flowering in Arabidopsis when overexpressed. Arabidopsis CDF genes are important negative regulators of CONSTANS (CO) transcription, but we found no effect of LATE2 on the transcription of pea CO-LIKE genes, nor on genes in any other families previously implicated in the activation of FT in Arabidopsis. Our results reveal an important component of the pea photoperiod response pathway and support the view that regulation of FTb2 expression by photoperiod occurs via a CO-independent mechanism.

Abscisic acid controlled sex before transpiration in vascular plants.

Sexual reproduction in animals and plants shares common elements, including sperm and egg production, but unlike animals, little is known about the regulatory pathways that determine the sex of plants. Here we use mutants and gene silencing in a fern species to identify a core regulatory mechanism in plant sexual differentiation. A key player in fern sex differentiation is the phytohormone abscisic acid (ABA), which regulates the sex ratio of male to hermaphrodite tissues during the reproductive cycle. Our analysis shows that in the fern Ceratopteris richardii, a gene homologous to core ABA transduction genes in flowering plants [SNF1-related kinase2s (SnRK2s)] is primarily responsible for the hormonal control of sex determination. Furthermore, we provide evidence that this ABA-SnRK2 signaling pathway has transitioned from determining the sex of ferns to controlling seed dormancy in the earliest seed plants before being co-opted to control transpiration and CO2 exchange in derived seed plants. By tracing the evolutionary history of this ABA signaling pathway from plant reproduction through to its role in the global regulation of plant-atmosphere gas exchange during the last 450 million years, we highlight the extraordinary effect of the ABA-SnRK2 signaling pathway in plant evolution and vegetation function.

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.

Leaves, not roots or floral tissue, are the main site of rapid, external pressure-induced ABA biosynthesis in angiosperms.

Rapid biosynthesis of abscisic acid (ABA) in the leaf, triggered by a decrease in cell volume, is essential for a functional stomatal response. However, it is not known whether rapid biosynthesis of ABA is also triggered in other plant tissues. Through the application of external pressure to flower, root, and leaf tissues, we test whether a reduction in cell volume can trigger rapid increases in ABA levels across the plant body in two species, Solanum lycopersicum and Passiflora tarminiana. Our results show that, in contrast to rapid ABA synthesis in the leaf, flower and root tissue did not show a significant, increase in ABA level in response to a drop in cell volume over a short time frame, suggesting that rapid ABA biosynthesis occurs only in leaf, not in flower or root tissues. A gene encoding the key, rate-limiting carotenoid cleavage enzyme (9-cis-epoxycarotenoid dioxygenase, NCED) in the ABA biosynthetic pathway in S. lycopersicum, NCED1, was upregulated to a lesser degree in flowers and roots compared with leaves in response to applied pressure. In both species, floral tissues contained substantially lower levels of the NCED substrate 9'-cis-neoxanthin than leaves, and this ABA precursor could not be detected in roots. Slow and minimal ABA biosynthesis was detected after 2 h in petals, indicating that floral tissue is capable of synthesizing ABA in response to sustained water deficit. Our results indicate that rapid ABA biosynthesis predominantly occurs in the leaves, and not in other tissues.

Pea VEGETATIVE2 Is an FD Homolog That Is Essential for Flowering and Compound Inflorescence Development.

As knowledge of the gene networks regulating inflorescence development in Arabidopsis thaliana improves, the current challenge is to characterize this system in different groups of crop species with different inflorescence architecture. Pea (Pisum sativum) has served as a model for development of the compound raceme, characteristic of many legume species, and in this study, we characterize the pea VEGETATIVE2 (VEG2) locus, showing that it is critical for regulation of flowering and inflorescence development and identifying it as a homolog of the bZIP transcription factor FD. Through detailed phenotypic characterizations of veg2 mutants, expression analyses, and the use of protein-protein interaction assays, we find that VEG2 has important roles during each stage of development of the pea compound inflorescence. Our results suggest that VEG2 acts in conjunction with multiple FLOWERING LOCUS T (FT) proteins to regulate expression of downstream target genes, including TERMINAL FLOWER1, LEAFY, and MADS box homologs, and to facilitate cross-regulation within the FT gene family. These findings further extend our understanding of the mechanisms underlying compound inflorescence development in pea and may have wider implications for future manipulation of inflorescence architecture in related legume crop species.

Up-regulation of NCED3 and ABA biosynthesis occur within minutes of a decrease in leaf turgor but AHK1 is not required.

A major environmental signal influencing day-time stomatal aperture is the vapour pressure deficit between the leaf and atmosphere (VPD). In angiosperms, increased VPD triggers biosynthesis of abscisic acid (ABA), prompting rapid stomatal closure. Altered cell turgor has been proposed as the trigger for ABA biosynthesis, but the timing and nature of the genetic signals linking these processes have remained uncertain. We investigated this in Arabidopsis by examining changes induced by a decrease in leaf turgor, simulating a natural increase in VPD. We found that the rate-limiting gene within the de novo ABA biosynthesis pathway, 9-cis-epoxycarotenoid dioxygenase 3 (NCED3), was induced and ABA levels increased within just 5 min of decreased leaf turgor. This rapid induction matches the time-frame for initiation of stomatal closure in response to a doubling in VPD. We further examined Arabidopsis histidine kinase1 (AHK1) as the most likely candidate for the turgor-sensing receptor involved, but found no significant difference between wild-type and an ahk1 null mutant in the induction of ABA-biosynthetic genes, ABA production, or stomatal behaviour. We show that decreased leaf turgor triggers de novo ABA biosynthesis within the time-frame of the stomatal response to VPD, but that AHK1 does not fulfil a critical role as a turgor-sensing receptor within this pathway.

What are the evolutionary origins of stomatal responses to abscisic acid in land plants?

The evolution of active stomatal closure in response to leaf water deficit, mediated by the hormone abscisic acid (ABA), has been the subject of recent debate. Two different models for the timing of the evolution of this response recur in the literature. A single-step model for stomatal control suggests that stomata evolved active, ABA-mediated control of stomatal aperture, when these structures first appeared, prior to the divergence of bryophyte and vascular plant lineages. In contrast, a gradualistic model for stomatal control proposes that the most basal vascular plant stomata responded passively to changes in leaf water status. This model suggests that active ABA-driven mechanisms for stomatal responses to water status instead evolved after the divergence of seed plants, culminating in the complex, ABA-mediated responses observed in modern angiosperms. Here we review the findings that form the basis for these two models, including recent work that provides critical molecular insights into resolving this intriguing debate, and find strong evidence to support a gradualistic model for stomatal evolution.

Stomatal responses to vapour pressure deficit are regulated by high speed gene expression in angiosperms.

Plants dynamically regulate water use by the movement of stomata on the surface of leaves. Stomatal responses to changes in vapour pressure deficit (VPD) are the principal regulator of daytime transpiration and water use efficiency in land plants. In angiosperms, stomatal responses to VPD appear to be regulated by the phytohormone abscisic acid (ABA), yet the origin of this ABA is controversial. After a 20 min exposure of plants, from three diverse angiosperm species, to a doubling in VPD, stomata closed, foliar ABA levels increased and the expression of the gene encoding the key, rate-limiting carotenoid cleavage enzyme (9-cis-epoxycarotenoid dioxygenase, NCED) in the ABA biosynthetic pathway was significantly up-regulated. The NCED gene was the only gene in the ABA biosynthetic pathway to be up-regulated over the short time scale corresponding to the response of stomata. The closure of stomata and rapid increase in foliar ABA levels could not be explained by the release of ABA from internal stores in the leaf or the hydrolysis of the conjugate ABA-glucose ester. These results implicate an extremely rapid de novo biosynthesis of ABA, mediated by a single gene, as the means by which angiosperm stomata respond to natural changes in VPD.

The Pea Photoperiod Response Gene STERILE NODES Is an Ortholog of LUX ARRHYTHMO.

The STERILE NODES (SN) locus in pea (Pisum sativum) was one of the first photoperiod response genes to be described and provided early evidence for the genetic control of long-distance signaling in flowering-time regulation. Lines homozygous for recessive sn mutations are early flowering and photoperiod insensitive, with an increased ability to promote flowering across a graft union in short-day conditions. Here, we show that SN controls developmental regulation of genes in the FT family and rhythmic regulation of genes related to circadian clock function. Using a positional and functional candidate approach, we identify SN as the pea ortholog of LUX ARRHYTHMO, a GARP transcription factor from Arabidopsis (Arabidopsis thaliana) with an important role in circadian clock function. In addition to induced mutants, sequence analysis demonstrates the presence of at least three other independent, naturally occurring loss-of-function mutations among known sn cultivars. Examination of genetic and regulatory interactions between SN and two other circadian clock genes, HIGH RESPONSE TO PHOTOPERIOD (HR) and DIE NEUTRALIS (DNE), suggests a complex relationship in which HR regulates expression of SN and the role of DNE and HR in control of flowering is dependent on SN. These results extend previous work to show that pea orthologs of all three Arabidopsis evening complex genes regulate clock function and photoperiod-responsive flowering and suggest that the function of these genes may be widely conserved.

The pea GIGAS gene is a FLOWERING LOCUS T homolog necessary for graft-transmissible specification of flowering but not for responsiveness to photoperiod.

Garden pea (Pisum sativum) was prominent in early studies investigating the genetic control of flowering and the role of mobile flowering signals. In view of recent evidence that genes in the FLOWERING LOCUS T (FT) family play an important role in generating mobile flowering signals, we isolated the FT gene family in pea and examined the regulation and function of its members. Comparison with Medicago truncatula and soybean (Glycine max) provides evidence of three ancient subclades (FTa, FTb, and FTc) likely to be common to most crop and model legumes. Pea FT genes show distinctly different expression patterns with respect to developmental timing, tissue specificity, and response to photoperiod and differ in their activity in transgenic Arabidopsis thaliana, suggesting they may have different functions. We show that the pea FTa1 gene corresponds to the GIGAS locus, which is essential for flowering under long-day conditions and promotes flowering under short-day conditions but is not required for photoperiod responsiveness. Grafting, expression, and double mutant analyses show that GIGAS/FTa1 regulates a mobile flowering stimulus but also provide clear evidence for a second mobile flowering stimulus that is correlated with expression of FTb2 in leaf tissue. These results suggest that induction of flowering by photoperiod in pea results from interactions among several members of a diversified FT family.