Ethylene controls three-dimensional growth involving reduced auxin levels in the moss Physcomitrium patens.

The conquest of land by plants was concomitant with, and possibly enabled by, the evolution of three-dimensional (3D) growth. The moss Physcomitrium patens provides a model system for elucidating molecular mechanisms in the initiation of 3D growth. Here, we investigate whether the phytohormone ethylene, which is believed to have been a signal before land plant emergence, plays a role in 3D growth regulation in P. patens. We report ethylene controls 3D gametophore formation, based on results from exogenously applied ethylene and genetic manipulation of PpEIN2, which is a central component in the ethylene signaling pathway. Overexpression (OE) of PpEIN2 activates ethylene responses and leads to earlier formation of gametophores with fewer gametophores produced thereafter, phenocopying ethylene-treated wild-type. Conversely, Ppein2 knockout mutants, which are ethylene insensitive, show initially delayed gametophore formation with more gametophores produced later. Furthermore, pharmacological and biochemical analyses reveal auxin levels are decreased in the OE lines but increased in the knockout mutants. Our results suggest that evolutionarily, ethylene and auxin molecular networks were recruited to build the plant body plan in ancestral land plants. This might have played a role in enabling ancient plants to acclimate to the continental surfaces of the planet.

A glutamate receptor-like gene is involved in ABA-mediated growth control in Physcomitrium (Physcomitrella) patens.

Plant glutamate receptor homologs (GLRs), which function as key calcium channels, play pivotal roles in various developmental processes as well as stress responses. The moss Physcomitrium patens, a representative of the earliest land plant lineage, possess multiple pathways of hormone signaling for coordinating growth and adaptation responses. However, it is not clear whether GLRs are connected to hormone-mediated growth control in the moss. In this study, we report that one of the two GLRs in P. patens, PpGLR1, involves in abscisic acid (ABA)-mediated growth regulation. ABA represses the growth of wild-type moss, and intriguingly, the PpGLR1 transcript levels are significantly increased in response to ABA treatment, based on both gene expression and the PpGLR1pro::GUS reporter results. Furthermore, the growth of Ppglr1 knockout moss mutants is hypersensitive to ABA treatment. These results suggest that PpGLR1 plays a critical role in ABA-mediated growth regulation, which provide useful information for our further investigation of the regulatory mechanism between Ca2+ signal and ABA in moss growth control.

Arabidopsis GLR3.5-modulated seed germination involves GA and ROS signaling.

Seed germination, a crucial developmental step, is regulated by multiple plant endogenous signals, among which phytohormones absisic acid (ABA) and gibberellin (GA) act antagonistically. Reactive oxygen species (ROS) interact with the two hormones to coordinate germination. We have previously reported that Arabidopsis glutamate receptor homolog3.5 (AtGLR3.5) modulates calcium signal to attenuate the repression effect of ABA on seed germination and that amino acid L-methionine functions upstream of AtGLR3.5, resulting in calcium influx. Here, we show that AtGLR3.5 modulates GA and ROS signaling during seed germination. Our findings provide a more complete picture as to the molecular mechanisms of AtGLR3.5 in seed germination control.

Identification of Quantitative Trait Loci Controlling Ethylene Production in Germinating Seeds in Maize (Zea mays L.).

Plant seed germination is a crucial developmental event that has significant effects on seedling establishment and yield production. This process is controlled by multiple intrinsic signals, particularly phytohormones. The gaseous hormone ethylene stimulates seed germination; however, the genetic basis of ethylene production in maize during seed germination remains poorly understood. In this study, we quantified the diversity of germination among 14 inbred lines representing the parental materials corresponding to multiple recombinant inbred line (RIL) mapping populations. Quantitative trait loci (QTLs) controlling ethylene production were then identified in germinating seeds from an RIL population constructed from two parental lines showing differences in both germination speed and ethylene production during germination. To explore the possible genetic correlations of ethylene production with other traits, seed germination and seed weight were evaluated using the same batch of samples. On the basis of high-density single nucleotide polymorphism-based genetic linkage maps, we detected three QTLs for ethylene production in germinating seeds, three QTLs for seed germination, and four QTLs for seed weight, with each QTL explaining 5.8%-13.2% of the phenotypic variation of the trait. No QTLs were observed to be co-localized, suggesting that the genetic bases underlying the three traits are largely different. Our findings reveal three chromosomal regions responsible for ethylene production during seed germination, and provide a valuable reference for the future investigation of the genetic mechanism underlying the role of the stress hormone ethylene in maize germination control under unfavourable external conditions.

Methionine synthase 1 provides methionine for activation of the GLR3.5 Ca2+ channel and regulation of germination in Arabidopsis.

Seed germination is a developmental process regulated by numerous internal and external cues. Our previous studies have shown that calcium influx mediated by the Arabidopsis glutamate receptor homolog 3.5 (AtGLR3.5) modulates the expression of the ABSCISIC ACID INSENSITIVE 4 (ABI4) transcription factor during germination and that L-methionine (L-Met) activates AtGLR3.1/3.5 Ca2+ channels in guard cells. However, it is not known whether L-Met participates in regulation of germination and what cellular mechanism is responsible for Met production during germination. Here, we describe Arabidopsis methionine synthase 1 (AtMS1), which acts in the final step of Met biosynthesis, synthesizes the Met required for the activation of AtGLR3.5 Ca2+ channels whose expression is up-regulated during germination, leading to the regulation of seed germination. We show that exogenous L-Met promotes germination in an AtGRL3.5-dependent manner. We also demonstrate that L-Met directly regulates the AtGLR3.5-mediated increase in cytosolic Ca2+ level in seedlings. We provide pharmacological and genetic evidence that Met synthesized via AtMS1 acts upstream of the AtGLR3.5-mediated Ca2+ signal and regulates the expression of ABI4, a major regulator in the abscisic acid response in seeds. Overall, our results link AtMS1, L-Met, the AtGLR3.5 Ca2+ channel, Ca2+ signals, and ABI4, and shed light on the physiological role and molecular mechanism of L-Met in germination.

A leucine-rich repeat-receptor-like kinase gene SbER2-1 from sorghum (Sorghum bicolor L.) confers drought tolerance in maize.

BACKGROUND: ERECTA (ER) is a leucine-rich repeat-receptor-like kinase gene (LRR-RLK) encoding a protein isolated from Arabidopsis. Although the regulatory functions of ER genes have been widely explored in plant development and disease resistance, their roles in drought stress responses remain to be clarified. RESULTS: In this study, we cloned and characterized two ER genes, SbER1-1 and SbER2-1, from the drought-tolerant model plant sorghum (Sorghum bicolor L.). Under drought stress, the two genes were expressed in the leaves and stems but not in the roots, and SbER2-1 transcript accumulation in the stem was increased. SbER2-1 was localized both on the plasma membrane and in the chloroplast. Moreover, SbER2-1 expression in Arabidopsis and maize conferred increased drought tolerance, especially in regard to water-use efficiency, increasing the net photosynthetic rate in maize under drought stress. Based on RNA-Seq analysis together with the physiological data, we conclude that the transgenic maize plants have upregulated phenylpropanoid metabolism and increased lignin accumulation under drought stress. CONCLUSIONS: Our results demonstrate that SbER2-1 plays an important role in response to drought stress. Furthermore, photosynthetic systems and phenylpropanoid metabolism are implicated in SbER2-1-mediated drought stress tolerance mechanisms. The use of genetic engineering to regulate SbER2-1 expression in plants and to breed new varieties tolerant to drought is a research field full of potential.

Genetic analysis of seedling root traits reveals the association of root trait with other agronomic traits in maize.

BACKGROUND: Root systems play important roles in crop growth and stress responses. Although genetic mechanism of root traits in maize (Zea mays L.) has been investigated in different mapping populations, root traits have rarely been utilized in breeding programs. Elucidation of the genetic basis of maize root traits and, more importantly, their connection to other agronomic trait(s), such as grain yield, may facilitate root trait manipulation and maize germplasm improvement. In this study, we analyzed genome-wide genetic loci for maize seedling root traits at three time-points after seed germination to identify chromosomal regions responsible for both seedling root traits and other agronomic traits in a recombinant inbred line (RIL) population (Zong3 × Yu87-1). RESULTS: Eight seedling root traits were examined at 4, 9, and 14 days after seed germination, and thirty-six putative quantitative trait loci (QTLs), accounting for 9.0-23.2% of the phenotypic variation in root traits, were detected. Co-localization of root trait QTLs was observed at, but not between, the three time-points. We identified strong or moderate correlations between root traits controlled by each co-localized QTL region. Furthermore, we identified an overlap in the QTL locations of seedling root traits examined here and six other traits reported previously in the same RIL population, including grain yield-related traits, plant height-related traits, and traits in relation to stress responses. Maize chromosomal bins 1.02-1.03, 1.07, 2.06-2.07, 5.05, 7.02-7.03, 9.04, and 10.06 were identified QTL hotspots for three or four more traits in addition to seedling root traits. CONCLUSIONS: Our identification of co-localization of root trait QTLs at, but not between, each of the three time-points suggests that maize seedling root traits are regulated by different sets of pleiotropic-effect QTLs at different developmental stages. Furthermore, the identification of QTL hotspots suggests the genetic association of seedling root traits with several other traits and reveals maize chromosomal regions valuable for marker-assisted selection to improve root systems and other agronomic traits simultaneously.

L-Met Activates Arabidopsis GLR Ca2+ Channels Upstream of ROS Production and Regulates Stomatal Movement.

Plant glutamate receptor homologs (GLRs) have long been proposed to function as ligand-gated Ca2+ channels, but no in planta evidence has been provided. Here, we present genetic evidence that Arabidopsis GLR3.1 and GLR3.5 form Ca2+ channels activated by L-methionine (L-Met) at physiological concentrations and regulate stomatal apertures and plant growth. The glr3.1/3.5 mutations resulted in a lower cytosolic Ca2+ level, defective Ca2+-induced stomatal closure, and Ca2+-deficient growth disorder, all of which involved L-Met. Patch-clamp analyses of guard cells showed that GLR3.1/3.5 Ca2+ channels are activated specifically by L-Met, with the activation abolished in glr3.1/3.5. Moreover, GLR3.1/3.5 Ca2+ channels are distinct from previously characterized ROS-activated Ca2+ channels and act upstream of ROS, providing Ca2+ transients necessary for the activation of NADPH oxidases. Our data indicate that GLR3.1/3.5 constitute L-Met-activated Ca2+ channels responsible for maintaining basal [Ca2+]cyt, play a pivotal role in plant growth, and act upstream of ROS, thereby regulating stomatal aperture.

Mechanistic Insights in Ethylene Perception and Signal Transduction.

The gaseous hormone ethylene profoundly affects plant growth, development, and stress responses. Ethylene perception occurs at the endoplasmic reticulum membrane, and signal transduction leads to a transcriptional cascade that initiates diverse responses, often in conjunction with other signals. Recent findings provide a more complete picture of the components and mechanisms in ethylene signaling, now rendering a more dynamic view of this conserved pathway. This includes newly identified protein-protein interactions at the endoplasmic reticulum membrane, as well as the major discoveries that the central regulator ETHYLENE INSENSITIVE2 (EIN2) is the long-sought phosphorylation substrate for the CONSTITUTIVE RESPONSE1 protein kinase, and that cleavage of EIN2 transmits the signal to the nucleus. In the nucleus, hundreds of potential gene targets of the EIN3 master transcription factor have been identified and found to be induced in transcriptional waves, and transcriptional coregulation has been shown to be a mechanism of ethylene cross talk.

Arabidopsis glutamate receptor homolog3.5 modulates cytosolic Ca2+ level to counteract effect of abscisic acid in seed germination.

Seed germination is a critical step in a plant's life cycle that allows successful propagation and is therefore strictly controlled by endogenous and environmental signals. However, the molecular mechanisms underlying germination control remain elusive. Here, we report that the Arabidopsis (Arabidopsis thaliana) glutamate receptor homolog3.5 (AtGLR3.5) is predominantly expressed in germinating seeds and increases cytosolic Ca2+ concentration that counteracts the effect of abscisic acid (ABA) to promote germination. Repression of AtGLR3.5 impairs cytosolic Ca2+ concentration elevation, significantly delays germination, and enhances ABA sensitivity in seeds, whereas overexpression of AtGLR3.5 results in earlier germination and reduced seed sensitivity to ABA. Furthermore, we show that Ca2+ suppresses the expression of ABSCISIC ACID INSENSITIVE4 (ABI4), a key transcription factor involved in ABA response in seeds, and that ABI4 plays a fundamental role in modulation of Ca2+-dependent germination. Taken together, our results provide molecular genetic evidence that AtGLR3.5-mediated Ca2+ influx stimulates seed germination by antagonizing the inhibitory effects of ABA through suppression of ABI4. These findings establish, to our knowledge, a new and pivotal role of the plant glutamate receptor homolog and Ca2+ signaling in germination control and uncover the orchestrated modulation of the AtGLR3.5-mediated Ca2+ signal and ABA signaling via ABI4 to fine-tune the crucial developmental process, germination, in Arabidopsis.

Conservation of ethylene as a plant hormone over 450 million years of evolution.

Land plants evolved more than 450 million years ago from a lineage of freshwater charophyte green algae(1). The extent to which plant signalling systems existed before the evolutionary transition to land is unknown. Although charophytes occupy a key phylogenetic position for elucidating the origins of such signalling systems(2-4), there is a paucity of sequence data for these organisms(5,6). Here we carry out de novo transcriptomics of five representative charophyte species, and find putative homologues for the biosynthesis, transport, perception and signalling of major plant hormones. Focusing on the plant hormone ethylene, we provide evidence that the filamentous charophyte Spirogyra pratensis possesses an ethylene hormone system homologous to that in plants. Spirogyra produces ethylene and exhibits a cell elongation response to ethylene. Spirogyra ethylene-signalling homologues partially rescue mutants of the angiosperm Arabidopsis thaliana and respond post-translationally to ethylene when expressed in plant cells, indicative of unambiguously homologous ethylene-signalling pathways in Spirogyra and Arabidopsis. These findings imply that the common aquatic ancestor possessed this pathway prior to the colonization of land and that cell elongation was possibly an ancestral ethylene response. This highlights the importance of charophytes for investigating the origins of fundamental plant processes.

Border control--a membrane-linked interactome of Arabidopsis.

Cellular membranes act as signaling platforms and control solute transport. Membrane receptors, transporters, and enzymes communicate with intracellular processes through protein-protein interactions. Using a split-ubiquitin yeast two-hybrid screen that covers a test-space of 6.4 × 10(6) pairs, we identified 12,102 membrane/signaling protein interactions from Arabidopsis. Besides confirmation of expected interactions such as heterotrimeric G protein subunit interactions and aquaporin oligomerization, >99% of the interactions were previously unknown. Interactions were confirmed at a rate of 32% in orthogonal in planta split-green flourescent protein interaction assays, which was statistically indistinguishable from the confirmation rate for known interactions collected from literature (38%). Regulatory associations in membrane protein trafficking, turnover, and phosphorylation include regulation of potassium channel activity through abscisic acid signaling, transporter activity by a WNK kinase, and a brassinolide receptor kinase by trafficking-related proteins. These examples underscore the utility of the membrane/signaling protein interaction network for gene discovery and hypothesis generation in plants and other organisms.

Advances in ethylene signalling: protein complexes at the endoplasmic reticulum membrane.

The gaseous plant hormone ethylene plays critical roles in plant responses to environmental and endogenous signals that modulate growth and development. Over the past 25 years, great progress has been made in elucidating the ethylene signalling pathway. Genetic studies in Arabidopsis thaliana have identified key components of the pathway, and subcellular localization studies have shown that most of these components, other than transcription factors and protein turnover machinery, are associated with or lie within the endoplasmic reticulum (ER) membrane. The ethylene receptors are found in high-molecular-mass protein complexes and interact with the CTR1 serine/threonine protein kinase and the genetically downstream EIN2 Nramp-like protein. To more fully understand the ethylene signalling pathway, recent research has focused on examining the molecular connections between these components and how they are regulated. Here, we review recent advances and remaining gaps in our understanding of the early steps in the ethylene signalling pathway taking place at the ER.

CTR1 phosphorylates the central regulator EIN2 to control ethylene hormone signaling from the ER membrane to the nucleus in Arabidopsis.

The gaseous phytohormone ethylene C(2)H(4) mediates numerous aspects of growth and development. Genetic analysis has identified a number of critical elements in ethylene signaling, but how these elements interact biochemically to transduce the signal from the ethylene receptor complex at the endoplasmic reticulum (ER) membrane to transcription factors in the nucleus is unknown. To close this gap in our understanding of the ethylene signaling pathway, the challenge has been to identify the target of the CONSTITUTIVE TRIPLE RESPONSE1 (CTR1) Raf-like protein kinase, as well as the molecular events surrounding ETHYLENE-INSENSITIVE2 (EIN2), an ER membrane-localized Nramp homolog that positively regulates ethylene responses. Here we demonstrate that CTR1 interacts with and directly phosphorylates the cytosolic C-terminal domain of EIN2. Mutations that block the EIN2 phosphorylation sites result in constitutive nuclear localization of the EIN2 C terminus, concomitant with constitutive activation of ethylene responses in Arabidopsis. Our results suggest that phosphorylation of EIN2 by CTR1 prevents EIN2 from signaling in the absence of ethylene, whereas inhibition of CTR1 upon ethylene perception is a signal for cleavage and nuclear localization of the EIN2 C terminus, allowing the ethylene signal to reach the downstream transcription factors. These findings significantly advance our understanding of the mechanisms underlying ethylene signal transduction.

The involvement of NtFtsZ2-1 gene in the regulation of chloroplast division and expansion in tobacco.

Chloroplasts are a vital group of organelles of plants, yet the molecular mechanisms associated with their division remain poorly understood. Recent studies have revealed that the FtsZ protein, known as a key component in prokaryotic cell division, is involved in chloroplast division process. The NtFtsZ2-1 gene was isolated from Nicotiana tabacum by RT-PCR, and the sense and antisense expression plasmids were used to examine the function of NtFtsZ2-1 gene in transgenic tobacco. Light and confocal observations revealed that the normal chloroplast division process was severely disrupted in transgenic plants with enhanced or reduced expression of NtFtsZ2-1 gene. These chloroplasts were abnormally larger in size and fewer in number compared with that of the wild-type tobacco. But the total chloroplast plan area per mesophyll cell was conserved in sense, antisense and wild type tobaccos. Analyses of electron micrographs and chlorophyll content of different transgenic plants showed that constitutively enhancing or inhibiting the expression of NtFtsZ2-1 gene had no direct influence on the ultrastructure and photosynthetic ability of chloroplasts. Basing on these results, we suggest that NtFtsZ2-1 gene is involved in chloroplast division and expansion; the fluctuation of NtFtsZ2-1 expression level would alter normal chloroplast number and size in plant cells. In addition, the similarities of ultrastructure and photosynthetic ability of chloroplasts among sense, antisense and wild type tobaccos implies that a special mechanism regulate the relationship between chloroplast number and size to maximize photosynthetic rate.

Cloning, chromosome mapping and expression analysis of an R2R3-MYB gene under-expressed in maize hybrid.

R2R3-MYB transcription factors play important role in transcriptional controls during higher plant metabolism and development. In this study, an R2R3-MYB gene was isolated from maize according to an EST, which expressed differentially between a hybrid and the two parents on a cDNA chip. The full-length cDNA, designated by ZmMYBL1 (GenBank accession no. AY365033) consists of 1417 nucleotides and contains an open reading frame of 828 bp. The deduced amino acid sequence contained two conserved MYB domains near its N-terminus, a conserved E1 motif and an acidic Ser/Thr rich region toward its C-terminus. Southern blot analysis revealed ZmMYBL1 could be a single copy gene belonging to a multi-gene family in the maize genome. Expression analysis showed ZmMYBL1 transcripts accumulated in various tissues examined, with strong level in tassel and weak level in leaf. Also it was under-expressed in root, stem, and leaf of hybrid as compared with that of the two parents. ZmMYBL1 was mapped on maize chromosome bin7.03 between two SSR markers, bn1g339 and umc1865 using Yuyu22 recombinant inbred line population. A QTL for root average diameter in maize seedlings was also localized on the corresponding region of chromosome 7 within the interval ZmMYBLI-bnIg1805. A possible role of ZmMYBL1 and its relation to maize heterosis were discussed based on these results.