The impact of heat stress in plant reproduction.

The increment in global temperature reduces crop productivity, which in turn threatens food security. Currently, most of our food supply is produced by plants and the human population is estimated to reach 9 billion by 2050. Gaining insights into how plants navigate heat stress in their reproductive phase is essential for effectively overseeing the future of agricultural productivity. The reproductive success of numerous plant species can be jeopardized by just one exceptionally hot day. While the effects of heat stress on seedlings germination and root development have been extensively investigated, studies on reproduction are limited. The intricate processes of gamete development and fertilization unfold within a brief timeframe, largely concealed within the flower. Nonetheless, heat stress is known to have important effects on reproduction. Considering that heat stress typically affects both male and female reproductive structures concurrently, it remains crucial to identify cultivars with thermotolerance. In such cultivars, ovules and pollen can successfully undergo development despite the challenges posed by heat stress, enabling the completion of the fertilization process and resulting in a robust seed yield. Hereby, we review the current understanding of the molecular mechanisms underlying plant resistance to abiotic heat stress, focusing on the reproductive process in the model systems of Arabidopsis and Oryza sativa.

Plant Ca2+-ATPases: From biochemistry to signalling.

Calcium (Ca2+)-ATPases are ATP-dependent enzymes that transport Ca2+ ions against their electrochemical gradient playing the fundamental biological function of keeping the free cytosolic Ca2+ concentration in the submicromolar range to prevent cytotoxic effects. In plants, type IIB autoinhibited Ca2+-ATPases (ACAs) are localised both at the plasma membrane and at the endomembranes including endoplasmic reticulum (ER) and tonoplast and their activity is primarily regulated by Ca2+-dependent mechanisms. Instead, type IIA ER-type Ca2+-ATPases (ECAs) are present mainly at the ER and Golgi Apparatus membranes and are active at resting Ca2+. Whereas research in plants has historically focused on the biochemical characterization of these pumps, more recently the attention has been also addressed on the physiological roles played by the different isoforms. This review aims to highlight the main biochemical properties of both type IIB and type IIA Ca2+ pumps and their involvement in the shaping of cellular Ca2+ dynamics induced by different stimuli.

Long-distance turgor pressure changes induce local activation of plant glutamate receptor-like channels.

In Arabidopsis thaliana, local wounding and herbivore feeding provoke leaf-to-leaf propagating Ca2+ waves that are dependent on the activity of members of the glutamate receptor-like channels (GLRs). In systemic tissues, GLRs are needed to sustain the synthesis of jasmonic acid (JA) with the subsequent activation of JA-dependent signaling response required for the plant acclimation to the perceived stress. Even though the role of GLRs is well established, the mechanism through which they are activated remains unclear. Here, we report that in vivo, the amino-acid-dependent activation of the AtGLR3.3 channel and systemic responses require a functional ligand-binding domain. By combining imaging and genetics, we show that leaf mechanical injury, such as wounds and burns, as well as hypo-osmotic stress in root cells, induces the systemic apoplastic increase of L-glutamate (L-Glu), which is largely independent of AtGLR3.3 that is instead required for systemic cytosolic Ca2+ elevation. Moreover, by using a bioelectronic approach, we show that the local release of minute concentrations of L-Glu in the leaf lamina fails to induce any long-distance Ca2+ waves.

Simultaneous imaging of ER and cytosolic Ca2+ dynamics reveals long-distance ER Ca2+ waves in plants.

Calcium ions (Ca2+) play a key role in cell signaling across organisms. In plants, a plethora of environmental and developmental stimuli induce specific Ca2+ increases in the cytosol as well as in different cellular compartments including the endoplasmic reticulum (ER). The ER represents an intracellular Ca2+ store that actively accumulates Ca2+ taken up from the cytosol. By exploiting state-of-the-art genetically encoded Ca2+ indicators, specifically the ER-GCaMP6-210 and R-GECO1, we report the generation and characterization of an Arabidopsis (Arabidopsis thaliana) line that allows for simultaneous imaging of Ca2+ dynamics in both the ER and cytosol at different spatial scales. By performing analyses in single cells, we precisely quantified (1) the time required by the ER to import Ca2+ from the cytosol into the lumen and (2) the time required to observe a cytosolic Ca2+ increase upon the pharmacological inhibition of the ER-localized P-Type IIA Ca2+-ATPases. Furthermore, live imaging of mature, soil-grown plants revealed the existence of a wounding-induced, long-distance ER Ca2+ wave propagating in injured and systemic rosette leaves. This technology enhances high-resolution analyses of intracellular Ca2+ dynamics at the cellular level and in adult organisms and paves the way to develop new methodologies aimed at defining the contribution of subcellular compartments in Ca2+ homeostasis and signaling.

A genetic approach reveals different modes of action of prefoldins.

The prefoldin complex (PFDc) was identified in humans as a co-chaperone of the cytosolic chaperonin T-COMPLEX PROTEIN RING COMPLEX (TRiC)/CHAPERONIN CONTAINING TCP-1 (CCT). PFDc is conserved in eukaryotes and is composed of subunits PFD1-6, and PFDc-TRiC/CCT folds actin and tubulins. PFDs also participate in a wide range of cellular processes, both in the cytoplasm and in the nucleus, and their malfunction causes developmental alterations and disease in animals and altered growth and environmental responses in yeast and plants. Genetic analyses in yeast indicate that not all of their functions require the canonical complex. The lack of systematic genetic analyses in plants and animals, however, makes it difficult to discern whether PFDs participate in a process as the canonical complex or in alternative configurations, which is necessary to understand their mode of action. To tackle this question, and on the premise that the canonical complex cannot be formed if one subunit is missing, we generated an Arabidopsis (Arabidopsis thaliana) mutant deficient in the six PFDs and compared various growth and environmental responses with those of the individual mutants. In this way, we demonstrate that the PFDc is required for seed germination, to delay flowering, or to respond to high salt stress or low temperature, whereas at least two PFDs redundantly attenuate the response to osmotic stress. A coexpression analysis of differentially expressed genes in the sextuple mutant identified several transcription factors, including ABA INSENSITIVE 5 (ABI5) and PHYTOCHROME-INTERACTING FACTOR 4, acting downstream of PFDs. Furthermore, the transcriptomic analysis allowed assigning additional roles for PFDs, for instance, in response to higher temperature.

The signatures of organellar calcium.

Recent insights about the transport mechanisms involved in the in and out of calcium ions in plant organelles, and their role in the regulation of cytosolic calcium homeostasis in different signaling pathways.

The Importance of Cytokinins during Reproductive Development in Arabidopsis and Beyond.

Fertilization and seed formation are fundamental events in the life cycle of flowering plants. The seed is a functional unit whose main purpose is to propagate the plant. The first step in seed development is the formation of male and female gametophytes and subsequent steps culminate in successful fertilization. The detailed study of this process is highly relevant because it directly impacts human needs, such as protecting biodiversity and ensuring sustainable agriculture to feed the increasing world population. Cytokinins comprise a class of phytohormones that play many important roles during plant growth and development and in recent years, the role of this class of phytohormones during reproduction has become clear. Here, we review the role of cytokinins during ovule, pollen and seed formation at the genetic and molecular levels. The expansion of knowledge concerning the molecular mechanisms that control plant reproduction is extremely important to optimise seed production.

Genetic Variation in Damaged Populations of Pistacia atlantica Desf.

The Atlas Pistachio tree, Pistacia atlantica Desf., has great importance in the ecological landscape of North Africa, due to its adaptive plasticity, as well as its use as a rootstock in the cultivation of the economically important species, Pistacia vera L. The conservation and valuation of this species require sampling and an assessment of its genetic variability. For the first time in North Africa, the inter-simple sequence repeats (ISSR) molecular marker has been used in genetic-diversity assessment and in the population relationships of P. atlantica subsp. atlantica. The ISSR markers tested showed 74.1% polymorphism, while molecular variance (AMOVA) analysis revealed a high percentage of the total genetic diversity of 55.7% among the four populations studied. Cluster analysis with neighbor-joining (NJ) and principal coordinate analysis (PCO) divided the study sites into four distinct groups according to their geographical locations (Tiaret, Batna, Djelfa, and Bechar). Isolation by distance or Mantel test gave a positive correlation of r = 0.86 between geographical and genetic distances. The results in this study indicate an absence of gene flow, implying that conservation efforts should be taken separately for each population.

Effects on Plant Growth and Reproduction of a Peach R2R3-MYB Transcription Factor Overexpressed in Tobacco.

In plants, anthocyanin production is controlled by MYB and bHLH transcription factors. In peach, among the members of these families, MYB10.1 and bHLH3 have been shown to be the most important genes for production of these pigments during fruit ripening. Anthocyanins are valuable molecules, and the overexpression of regulatory genes in annual fast-growing plants has been explored for their biotechnological production. The overexpression of peach MYB10.1 in tobacco plants induced anthocyanin pigmentation, which was particularly strong in the reproductive parts. Pigment production was the result of an up-regulation of the expression level of key genes of the flavonoid biosynthetic pathway, such as NtCHS, NtCHI, NtF3H, NtDFR, NtANS, and NtUFGT, as well as of the proanthocyanidin biosynthetic pathway such as NtLAR. Nevertheless, phenotypic alterations in transgenic tobacco lines were not only limited to anthocyanin production. Lines showing a strong phenotype (type I) exhibited irregular leaf shape and size and reduced plant height. Moreover, flowers had reduced length of anther's filament, nondehiscent anthers, reduced pistil length, aborted nectary glands, and impaired capsule development, but the reproductive parts including androecium, gynoecium, and petals were more pigmented that in wild type. Surprisingly, overexpression of peach MYB10.1 led to suppression of NtMYB305, which is required for floral development and, of one of its target genes, NECTARIN1 (NtNCE1), involved in the nectary gland formation. MYB10.1 overexpression up-regulated JA biosynthetic (NtAOS) and signaling (NtJAZd) genes, as well as 1-aminocyclopropane-1-carboxylate oxidase (NtACO) in flowers. The alteration of these hormonal pathways might be among the causes of the observed floral abnormalities with defects in both male and female gametophyte development. In particular, approximately only 30% of pollen grains of type I lines were viable, while during megaspore formation, there was a block during FG1 (St3-II). This block seemed to be associated to an excessive accumulation of callose. It can be concluded that the overexpression of peach MYB10.1 in tobacco not only regulates flavonoid biosynthesis (anthocyanin and proanthocyanidin) in the reproductive parts but also plays a role in other processes such as vegetative and reproductive development.

The MPK8-TCP14 pathway promotes seed germination in Arabidopsis.

The accurate control of dormancy release and germination is critical for successful plantlet establishment. Investigations in cereals hypothesized a crucial role for specific MAP kinase (MPK) pathways in promoting dormancy release, although the identity of the MPK involved and the downstream events remain unclear. In this work, we characterized mutants for Arabidopsis thaliana MAP kinase 8 (MPK8). Mpk8 seeds presented a deeper dormancy than wild-type (WT) at harvest that was less efficiently alleviated by after-ripening and gibberellic acid treatment. We identified Teosinte Branched1/Cycloidea/Proliferating cell factor 14 (TCP14), a transcription factor regulating germination, as a partner of MPK8. Mpk8 tcp14 double-mutant seeds presented a deeper dormancy at harvest than WT and mpk8, but similar to that of tcp14 seeds. MPK8 interacted with TCP14 in the nucleus in vivo and phosphorylated TCP14 in vitro. Furthermore, MPK8 enhanced TCP14 transcriptional activity when co-expressed in tobacco leaves. Nevertheless, the stimulation of TCP14 transcriptional activity by MPK8 could occur independently of TCP14 phosphorylation. The comparison of WT, mpk8 and tcp14 transcriptomes evidenced that whereas no effect was observed in dry seeds, mpk8 and tcp14 mutants presented dramatic transcriptomic alterations after imbibition with a sustained expression of genes related to seed maturation. Moreover, both mutants exhibited repression of genes involved in cell wall remodeling and cell cycle G1/S transition. As a whole, this study unraveled a role for MPK8 in promoting seed germination, and suggested that its interaction with TCP14 was critical for regulating key processes required for germination completion.

Time-Course Transcriptome Analysis of Arabidopsis Siliques Discloses Genes Essential for Fruit Development and Maturation.

Fruits protect the developing seeds of angiosperms and actively contribute to seed dispersion. Furthermore, fruit and seed development are highly synchronized and require exchange of information between the mother plant and the developing generations. To explore the mechanisms controlling fruit formation and maturation, we performed a transcriptomic analysis on the valve tissue of the Arabidopsis (Arabidopsis thaliana) silique using RNA sequencing. In doing so, we have generated a data set of differentially regulated genes that will help to elucidate the molecular mechanisms that underpin the initial phase of fruit growth and, subsequently, trigger fruit maturation. The robustness of our data set has been tested by functional genomic studies. Using a reverse genetics approach, we selected 10 differentially expressed genes and explored the consequences of their disruption for both silique growth and senescence. We found that genes contained in our data set play essential roles in different stages of silique development and maturation, indicating that our transcriptome-based gene list is a powerful tool for the elucidation of the molecular mechanisms controlling fruit formation in Arabidopsis.

Trans-splicing of plastid rps12 transcripts, mediated by AtPPR4, is essential for embryo patterning in Arabidopsis thaliana.

MAIN CONCLUSION: AtPPR4-mediated trans-splicing of plastid rps12 transcripts is essential for key embryo morphogenetic events such as development of cotyledons, determination of provascular tissue, and organization of the shoot apical meristem (SAM), but not for the formation of the protodermal layer. Members of the pentatricopeptide repeat (PPR) containing protein family have emerged as key regulators of the organelle post-transcriptional processing and to be essential for proper plant embryo development. In this study, we report the functional characterization of the AtPPR4 (At5g04810) gene encoding a plastid nucleoid PPR protein. In-situ hybridization analysis reveals the presence of AtPPR4 transcripts already at the transition stage of embryo development. As a consequence, embryos lacking the AtPPR4 protein arrest their development at the transition/early-heart stages and show defects in the determination of the provascular tissue and organization of SAM. This complex phenotype is due to the specific role of AtPPR4 in the trans-splicing of the plastid rps12 transcripts, as shown by northern and slot-blot hybridizations, and the consequent defect in 70S ribosome accumulation and plastid protein synthesis, in agreement with the role proposed for the maize orthologue, ZmPPR4.

The Peach RGF/GLV Signaling Peptide pCTG134 Is Involved in a Regulatory Circuit That Sustains Auxin and Ethylene Actions.

In vascular plants the cell-to-cell interactions coordinating morphogenetic and physiological processes are mediated, among others, by the action of hormones, among which also short mobile peptides were recognized to have roles as signals. Such peptide hormones (PHs) are involved in defense responses, shoot and root growth, meristem homeostasis, organ abscission, nutrient signaling, hormone crosstalk and other developmental processes and act as both short and long distant ligands. In this work, the function of CTG134, a peach gene encoding a ROOT GROWTH FACTOR/GOLVEN-like PH expressed in mesocarp at the onset of ripening, was investigated for its role in mediating an auxin-ethylene crosstalk. In peach fruit, where an auxin-ethylene crosstalk mechanism is necessary to support climacteric ethylene synthesis, CTG134 expression peaked before that of ACS1 and was induced by auxin and 1-methylcyclopropene (1-MCP) treatments, whereas it was minimally affected by ethylene. In addition, the promoter of CTG134 fused with the GUS reporter highlighted activity in plant parts in which the auxin-ethylene interplay is known to occur. Arabidopsis and tobacco plants overexpressing CTG134 showed abnormal root hair growth, similar to wild-type plants treated with a synthetic form of the sulfated peptide. Moreover, in tobacco, lateral root emergence and capsule size were also affected. In Arabidopsis overexpressing lines, molecular surveys demonstrated an impaired hormonal crosstalk, resulting in a re-modulated expression of a set of genes involved in both ethylene and auxin synthesis, transport and perception. These data support the role of pCTG134 as a mediator in an auxin-ethylene regulatory circuit and open the possibility to exploit this class of ligands for the rational design of new and environmental friendly agrochemicals able to cope with a rapidly changing environment.

SUPPRESSOR OF FRIGIDA (SUF4) Supports Gamete Fusion via Regulating Arabidopsis EC1 Gene Expression.

The EGG CELL1 (EC1) gene family of Arabidopsis (Arabidopsis thaliana) comprises five members that are specifically expressed in the egg cell and redundantly control gamete fusion during double fertilization. We investigated the activity of all five EC1 promoters in promoter-deletion studies and identified SUF4 (SUPPRESSOR OF FRIGIDA4), a C2H2 transcription factor, as a direct regulator of the EC1 gene expression. In particular, we demonstrated that SUF4 binds to all five Arabidopsis EC1 promoters, thus regulating their expression. The down-regulation of SUF4 in homozygous suf4-1 ovules results in reduced EC1 expression and delayed sperm fusion, which can be rescued by expressing SUF4-ß-glucuronidase under the control of the SUF4 promoter. To identify more gene products able to regulate EC1 expression together with SUF4, we performed coexpression studies that led to the identification of MOM1 (MORPHEUS' MOLECULE1), a component of a silencing mechanism that is independent of DNA methylation marks. In mom1-3 ovules, both SUF4 and EC1 genes are down-regulated, and EC1 genes show higher levels of histone 3 lysine-9 acetylation, suggesting that MOM1 contributes to the regulation of SUF4 and EC1 gene expression.

Characterization of TM8, a MADS-box gene expressed in tomato flowers.

BACKGROUND: The identity of flower organs is specified by various MIKC MADS-box transcription factors which act in a combinatorial manner. TM8 is a MADS-box gene that was isolated from the floral meristem of a tomato mutant more than twenty years ago, but is still poorly known from a functional point of view in spite of being present in both Angiosperms and Gymnosperms, with some species harbouring more than one copy of the gene. This study reports a characterization of TM8 that was carried out in transgenic tomato plants with altered expression of the gene. RESULTS: Tomato plants over-expressing either TM8 or a chimeric repressor form of the gene (TM8:SRDX) were prepared. In the TM8 up-regulated plants it was possible to observe anomalous stamens with poorly viable pollen and altered expression of several floral identity genes, among them B-, C- and E-function ones, while no apparent morphological modifications were visible in the other whorls. Oblong ovaries and fruits, that were also parthenocarpic, were obtained in the plants expressing the TM8:SRDX repressor gene. Such ovaries showed modified expression of various carpel-related genes. No apparent modifications could be seen in the other flower whorls. The latter plants had also epinastic leaves and malformed flower abscission zones. By using yeast two hybrid assays it was possible to show that TM8 was able to interact in yeast with MACROCALIX. CONCLUSIONS: The impact of the ectopically altered TM8 expression on the reproductive structures suggests that this gene plays some role in the development of the tomato flower. MACROCALYX, a putative A-function MADS-box gene, was expressed in all the four whorls of fully developed flowers, and showed quantitative variations that were opposite to those of TM8 in the anomalous stamens and ovaries. Since the TM8 protein interacted in vitro only with the A-function MADS-box protein MACROCALYX, it seems that for the correct differentiation of the tomato reproductive structures possible interactions between TM8 and MACROCALYX proteins might be important.