Hybrid Proline-rich Protein Research Articles

CRISPR-Cas9-based precise engineering of SlHyPRP1 protein towards multi-stress tolerance in tomato.

Recently, CRISPR-Cas9-based genome editing has been widely used for plant breeding. In our previous report, a tomato gene encoding hybrid proline-rich protein 1 (HyPRP1), a negative regulator of salt stress responses, has been edited using a CRISPR-Cas9 multiplexing approach that resulted in precise eliminations of its functional domains, proline-rich domain (PRD) and eight cysteine-motif (8CM). We subsequently demonstrated that eliminating the PRD domain of HyPRP1 in tomatoes conferred the highest level of salinity tolerance. In this study, we characterized the edited lines under several abiotic and biotic stresses to examine the possibility of multiple stress tolerance. Our data reveal that the 8CM removal variants of HK and the KO alleles of both HK and 15T01 cultivars exhibited moderate heat stress tolerance. Similarly, plants carrying either the domains of the PRD removal variant (PR1v1) or 8CM removal variants (PR2v2 and PR2v3) showed better germination under osmosis stress (up to 200 mM mannitol) compared to the WT control. Moreover, the PR1v1 line continuously grew after 5 days of water cutoff. When the edited lines were challenged with pathogenic bacteria of Pseudomonas syringae pv. tomato (Pto) DC3000, the growth of the bacterium was significantly reduced by 2.0- to 2.5-fold compared to that in WT plants. However, the edited alleles enhanced susceptibility against Fusarium oxysporum f. sp. lycopersici, which causes fusarium wilt. CRISPR-Cas9-based precise domain editing of the SlHyPRP1 gene generated multi-stress-tolerant alleles that could be used as genetic materials for tomato breeding.

Stress responsive OsHyPRP16 promoter driven early expression of resistance gene Pi54 potentiate the resistance against Magnaporthe oryzae in transgenic rice

The rice Hybrid Proline Rich Protein (HyPRP) encoding gene, OsHyPRP16 expression exhibit early upregulation in response to Magnaporthe oryzae inoculation. Here, we functionally characterized the OsHyPRP16 promoter through deletion analysis in transgenic Arabidopsis using GUS (β-glucuronidase) reporter assay. The promoter fragments, sequentially deleted from the 5′ end could induce differential GUS activity in response to stresses induced by different hormones and abiotic stress conditions. In addition, a strong GUS induction was observed in M. oryzae inoculated transgenic Arabidopsis. Based on the insilico and stress-inducibility of D1 promoter fragment against various phytohormones and rice blast fungus, and with no basal activity under control conditions, we rationally selected D1 promoter fragment to drive the expression of a major rice blast resistance gene; Pi54 in the genetic background of blast susceptible TP309 rice line. The D1 promoter fragment was able to induce the expression of Pi54 at immediate-early stages of M. oryzae infection in transgenic rice. The transgenic plants with Pi54 under the control of D1 promoter fragment displayed complete resistance against M. oryzae infection as compared to control plants. The present study suggests that the D1 fragment of OsHyPRP16 promoter is a valuable tool for breeding and development of rice lines with early-inducible and pathogen-responsive enhanced disease resistance.

Friend or foe: Hybrid proline-rich proteins determine how plants respond to beneficial and pathogenic microbes.

Plant plastids generate signals, including some derived from lipids, that need to be mobilized to effect signaling. We used informatics to discover potential plastid membrane proteins involved in microbial responses in Arabidopsis (Arabidopsis thaliana). Among these are proteins co-regulated with the systemic immunity component AZELAIC ACID INDUCED 1, a hybrid proline-rich protein (HyPRP), and HyPRP superfamily members. HyPRPs have a transmembrane domain, a proline-rich region (PRR), and a lipid transfer protein domain. The precise subcellular location(s) and function(s) are unknown for most HyPRP family members. As predicted by informatics, a subset of HyPRPs has a pool of proteins that target plastid outer envelope membranes via a mechanism that requires the PRR. Additionally, two HyPRPs may be associated with thylakoid membranes. Most of the plastid- and nonplastid-localized family members also have pools that localize to the endoplasmic reticulum, plasma membrane, or plasmodesmata. HyPRPs with plastid pools regulate, positively or negatively, systemic immunity against the pathogen Pseudomonas syringae. HyPRPs also regulate the interaction with the plant growth-promoting rhizobacteria Pseudomonas simiae WCS417 in the roots to influence colonization, root system architecture, and/or biomass. Thus, HyPRPs have broad and distinct roles in immunity, development, and growth responses to microbes and reside at sites that may facilitate signal molecule transport.

CRISPR/Cas9-based precise excision of SlHyPRP1 domain(s) to obtain salt stress-tolerant tomato.

CRISPR/Cas9-based multiplexed editing of SlHyPRP1 resulted in precise deletions of its functional motif(s), thereby resulting in salt stress-tolerant events in cultivated tomato. Crop genetic improvement to address environmental stresses for sustainable food production has been in high demand, especially given the current situation of global climate changes and reduction of the global food production rate/population rate. Recently, the emerging clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas)-based targeted mutagenesis has provided a revolutionary approach to crop improvement. The major application of CRISPR/Cas in plant genome editing has been the generation of indel mutations via error-prone nonhomologous end joining (NHEJ) repair of DNA DSBs. In this study, we examined the power of the CRISPR/Cas9-based novel approach in the precise manipulation of protein domains of tomato hybrid proline-rich protein 1 (HyPRP1), which is a negative regulator of salt stress responses. We revealed that the precise elimination of SlHyPRP1 negative-response domain(s) led to high salinity tolerance at the germination and vegetative stages in our experimental conditions. CRISPR/Cas9-based domain editing may be an efficient tool to engineer multidomain proteins of important food crops to cope with global climate changes for sustainable agriculture and future food security.

Regulatory function of EARLI1-LIKE HYBRID PROLINE-RICH PROTEIN 1 in the floral transition of Arabidopsis thaliana

The plasma membrane- and endoplasmic reticulum-localized proteins encoded by the EARLI1 (EARLY ARABIDOPSIS ALUMINUM INDUCED 1) subfamily of genes contain a signal peptide, a proline-rich domain and an eight-cysteine motif. We previously showed that knockdown of EARLI1 subfamily genes in the late-flowering background of Col-FRI-Sf2 accelerates flowering time, suggesting that EARLI1 genes participate in maintenance of vegetative growth and prevention of premature reproductive growth. We show here that ELHyPRP1 (EARLI1-LIKE HYBRID PROLINE-RICH PROTEIN 1, AT4G12490), a member of this subfamily, is induced by environmental factors such as cold and long-day photoperiod. Detailed analyses with RNA interference and overexpressing lines demonstrated that modulation of ELHyPRP1 can affect the expression of photoperiod, vernalization, gibberellin, age, and autonomous flowering pathway genes. When ELHyPRP1 and other EARLI1 subfamily genes were silenced by RNA interference, most genes participating in the promotion of flowering were activated, while suppressors of the floral transition were repressed. Downregulation of ELHyPRP1 leads to increased expression of the flowering time genes FT and SOC1, and enhanced circadian expression patterns of genes associated with the promotion of the floral transition. Together with the silencing of ELHyPRP1, the flowering repressor FLC and the GA signalling repressor RGA were inhibited. ELHyPRP1 transgenic tobacco plants displayed late-flowering phenotypes and their flowering time was remarkably delayed in comparison to the wild-type Qinyan 95 tobacco plants grown under long-day photoperiod. These results indicate that ELHyPRP1 may be involved in balancing biomass accumulation and seed proliferation.

The Tomato Hybrid Proline-rich Protein regulates the abscission zone competence to respond to ethylene signals

The Tomato Hybrid Proline-rich Protein (THyPRP) gene was specifically expressed in the tomato (Solanum lycopersicum) flower abscission zone (FAZ), and its stable antisense silencing under the control of an abscission zone (AZ)-specific promoter, Tomato Abscission Polygalacturonase4, significantly inhibited tomato pedicel abscission following flower removal. For understanding the THyPRP role in regulating pedicel abscission, a transcriptomic analysis of the FAZ of THyPRP-silenced plants was performed, using a newly developed AZ-specific tomato microarray chip. Decreased expression of THyPRP in the silenced plants was already observed before abscission induction, resulting in FAZ-specific altered gene expression of transcription factors, epigenetic modifiers, post-translational regulators, and transporters. Our data demonstrate that the effect of THyPRP silencing on pedicel abscission was not mediated by its effect on auxin balance, but by decreased ethylene biosynthesis and response. Additionally, THyPRP silencing revealed new players, which were demonstrated for the first time to be involved in regulating pedicel abscission processes. These include: gibberellin perception, Ca2+-Calmodulin signaling, Serpins and Small Ubiquitin-related Modifier proteins involved in post-translational modifications, Synthaxin and SNARE-like proteins, which participate in exocytosis, a process necessary for cell separation. These changes, occurring in the silenced plants early after flower removal, inhibited and/or delayed the acquisition of the competence of the FAZ cells to respond to ethylene signaling. Our results suggest that THyPRP acts as a master regulator of flower abscission in tomato, predominantly by playing a role in the regulation of the FAZ cell competence to respond to ethylene signals.

A hybrid proline-rich cell-wall protein gene JsPRP1 from Juglans sigillata Dode confers both biotic and abiotic stresses in transgenic tobacco plants

Walnut hybrid proline-rich protein gene JsPRP1 was induced by Colletotrichum gloeosporioides and signaling molecules. JsPRP1 protein inhibited pathogenic fungi. JsPRP1 overexpression in tobacco conferred strong resistance to abiotic and biotic stresses. Walnut (Juglans sigillata Dode) is one of the important sources of nuts and timbers in the world. With the expansion of walnut cultivation area, its disease hazards have become a serious problem. In this study, a novel hybrid proline-rich cell-wall protein gene JsPRP1 was investigated and isolated from Juglans sigillata Dode induced by Colletotrichum gloeosporioides. qRT-PCR (quantitative reverse transcription-PCR) analysis showed that JsPRP1 transcriptional level was up-regulated in J. sigillata Dode following jasmonic acid, salicylic acid, ethylene, and hydrogen peroxide treatments, and C. gloeosporioides inoculation. The JsPRP1 gene was further fused with green fluorescent protein (GFP) gene in a plant expression vector and transferred into onion (Allium cepa) epidermal cells. Laser scanning confocal microscope confirmed that JsPRP1 was expressed in the cell wall. Moreover, the JsPRP1 protein was expressed in Escherichia coli, and the recombinant protein purified by Ni–NTA column showed in vitro antifungal activity to C. gloeosporioides, Gibberella moniliformis, Botryosphaeria dothidea, and Fusarium solani. Furthermore, the plant overexpression vector of JsPRP1 was constructed and transferred into tobacco. The T2 transgenic tobacco showed high tolerance to drought, CdCl2, and C. gloeosporioides infection, meanwhile the accumulation of reactive oxygen species (ROS) in transgenic tobacco was significantly lower than wild-type plant under stress. The above results indicate that JsPRP1 is an important defense gene in J. sigillata Dode against drought, CdCl2 stress, and C. gloeosporioides infection.

HyPRP1 Gene Suppressed by Multiple Stresses Plays a Negative Role in Abiotic Stress Tolerance in Tomato

Many hybrid proline-rich protein (HyPRP) genes respond to biotic and abiotic stresses in plants, but little is known about their roles other than as putative cell-wall structural proteins. A HyPRP1 gene encodes a protein with proline-rich domain, and an eight-cysteine motif was identified from our previous microarray experiments on drought-tolerant tomato. In this study, the expression of the HyPRP1 gene in tomato was suppressed under various abiotic stresses, such as drought, high salinity, cold, heat, and oxidative stress. Transgenic functional analysis showed no obvious changes in phenotypes, but enhanced tolerance to various abiotic stresses (e.g., oxidative stress, dehydration, and salinity) was observed in RNAi transgenic plants. Interestingly, several SO2 detoxification-related enzymes, including sulfite oxidase, ferredoxins (Fds), and methionine sulfoxide reductase A (Msr A), were revealed in HyPRP1-interacting proteins identified by Yeast Two-Hybrid screening. More sulfates and transcripts of Msr A and Fds were accumulated in HyPRP1 knockdown lines when wild-type plants were exposed to SO2 gas. Our findings illustrate that the tomato HyPRP1 is a negative regulator of salt and oxidative stresses and is probably involved in sulfite metabolism.

Post-Translational Modification and Secretion of Azelaic Acid Induced 1 (AZI1), a Hybrid Proline-Rich Protein from Arabidopsis.

Arabidopsis EARLI-type hybrid proline-rich proteins (HyPRPs) consist of a putative N-terminal secretion signal, a proline-rich domain (PRD), and a characteristic eight-cysteine-motif (8-CM). They have been implicated in biotic and abiotic stress responses. AZI1 is required for systemic acquired resistance and it has recently been identified as a target of the stress-induced mitogen-activated protein kinase MPK3. AZI1 gel migration properties strongly indicate AZI1 to undergo major post-translational modifications. These occur in a stress-independent manner and are unrelated to phosphorylation by MAPKs. As revealed by transient expression of AZI1 in Nicotiana benthamiana and Tropaeolum majus, the Arabidopsis protein is similarly modified in heterologous plant species. Proline-rich regions, resembling arabinogalactan proteins point to a possible proline hydroxylation and subsequent O-glycosylation of AZI1. Consistently, inhibition of prolyl hydroxylase reduces its apparent protein size. AZI1 secretion was examined using Arabidopsis protoplasts and seedling exudates. Employing Agrobacterium-mediated leaf infiltration of N. benthamiana, we attempted to assess long-distance movement of AZI1. In summary, the data point to AZI1 being a partially secreted protein and a likely new member of the group of hydroxyproline-rich glycoproteins. Its dual location suggests AZI1 to exert both intra- and extracellular functions.

A novel hybrid proline-rich type gene GsEARLI17 from Glycine soja participated in leaf cuticle synthesis and plant tolerance to salt and alkali stresses

Recent discoveries showed that hybrid proline rich type proteins were involved in plant responses to several environmental stresses. However, little is known about their roles in plant alkali stress adaptation, especially in wild soybean. Here, we isolated and characterized GsEARLI17, a novel the hybrid proline-rich protein type EARLI family gene from Glycine soja. Bioinformatic analysis showed that GsEARLI17 protein contained a conserved N-terminus hybrid proline-rich domain in and a C-terminus 8CM domain. Transcript abundance of GsEARLI17 was higher in young tissues than that in old tissues, and induced following exposure to salt and alkali treatments. GsEARLI17 overexpression in Arabidopsis influenced cuticles formation, as evidenced by thicker cuticles of transgenic lines, and enhanced plant tolerance to salt stress. Further observation was found that compared to the wild type Arabidopsis, overexpression of GsEARLI17 in Arabidopsis improved seed germination with higher leaf opening and greening rate during the early stages under alkali stress. GsEARLI17 exhibited enhanced alkaline tolerance with higher chlorophyll content, and lower malondialdehyde content at the adult developmental stage, and the expression levels of some alkali stress response marker genes NADP-ME, H + Ppase were higher in the GsEARLI17 overexpression lines than in wild-type plants. Taken together we suggest that GsEARLI17 reveal a positive role in enhancing plant tolerance of alkali stress in Arabidopsis.

Salt Stress in Arabidopsis: Lipid Transfer Protein AZI1 and Its Control by Mitogen-Activated Protein Kinase MPK3

A plant's capability to cope with environmental challenges largely relies on signal transmission through mitogen-activated protein kinase (MAPK) cascades. In Arabidopsis thaliana, MPK3 is particularly strongly associated with numerous abiotic and biotic stress responses. Identification of MPK3 substrates is a milestone towards improving stress resistance in plants. Here, we characterize AZI1, a lipid transfer protein (LTP)-related hybrid proline-rich protein (HyPRP), as a novel target of MPK3. AZI1 is phosphorylated by MPK3 in vitro. As documented by co-immunoprecipitation and bimolecular fluorescence complementation experiments, AZI1 interacts with MPK3 to form protein complexes in planta. Furthermore, null mutants of azi1 are hypersensitive to salt stress, while AZI1-overexpressing lines are markedly more tolerant. AZI1 overexpression in the mpk3 genetic background partially alleviates the salt-hypersensitive phenotype of this mutant, but functional MPK3 appears to be required for the full extent of AZI1-conferred robustness. Notably, this robustness does not come at the expense of normal development. Immunoblot and RT-PCR data point to a role of MPK3 as positive regulator of AZI1 abundance.

Plasmalemma localisation of DOUBLE HYBRID PROLINE-RICH PROTEIN 1 and its function in systemic acquired resistance of Arabidopsis thaliana.

The protein encoded by AtDHyPRP1 (DOUBLE HYBRID PROLINE-RICH PROTEIN 1) contains two tandem PRD-8CMs (proline-rich domain-eight cysteine motif) and represents a new type of HyPRPs (hybrid proline-rich proteins). Confocal microscopy to transgenic Arabidopsis plants revealed that AtDHyPRP1-GFP was localised to plasmalemma, especially plasmodesmata. AtDHyPRP1 mainly expressed in leaf tissues and could be induced by salicylic acid, methyl jasmonate, virulent Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) and avirulent P. syringae pv. tomato DC3000 harbouring avrRPM1 (Pst avrRPM1), suggesting it is involved in defence response of Arabidopsis thaliana (L. Heynh.). After treatments with bacterial suspension of virulent Pst DC3000 or conidial suspension of Botrytis cinerea, AtDHyPRP1 overexpressing lines exhibited enhanced resistance, whereas AtDHyPRP1 RNA interference lines became more susceptible to the pathogens with obvious chlorosis or necrosis phenotypes. In systemic acquired resistance (SAR) analyses, distal leaves were challenged with virulent Pst DC3000 after inoculation of the primary leaves with avirulent Pst avrRPM1 (AV) or MgSO4 (MV). Compared with MV, the infection symptoms in systemic leaves of wild-type plants and AtDHyPRP1 overexpressing lines were significantly alleviated in AV treatment, whereas the systemic leaves of AtDHyPRP1 RNAi lines were vulnerable to Pst DC3000, indicating AtDHyPRP1 was functionally associated with SAR.

Cotton GhHyPRP3 encoding a hybrid proline-rich protein is stress inducible and its overexpression in Arabidopsis enhances germination under cold temperature and high salinity stress conditions

In this study, the cDNA coding for a hybrid proline-rich protein (HyPRP) was isolated from cotton cDNA libraries and designated GhHyPRP3. Analysis of the deduced amino acid sequence revealed that it contained an N-terminal signal peptide, a central proline-rich domain, and a C-terminal cysteine-rich domain highly homologous to other hybrid proline-rich group B proteins. RNA gel blot analysis showed that GhHyPRP3 mRNA was most abundant in petals and 10 DPA ovules indicating that expression of GhHyPRP3 was petal-preferential and ovule developmentally regulated. In addition, GhHyPRP3 transcription in roots was up-regulated by salt stress, cold stress, and osmotic stress, but down-regulated by GA3. A promoter-GUS reporter revealed that the GhHyPRP3 promoter directed gene expression in root–shoot junction, roots, and petals of transgenic Arabidopsis plants. Subcellular localization results showed that GhHyPRP3 was localized to the plasma membrane. Transgenic lines overexpressing GhHyPRP3 had a higher germination rate under cold temperature and high salinity stress conditions compared with wild type. Overall, GhHyPRP3 may function in flower and ovule development and participate in the defense responses to low temperature and salt stress.

Expression of recombinant EARLI1, a hybrid proline-rich protein of Arabidopsis, in Escherichia coli and its inhibition effect to the growth of fungal pathogens and Saccharomyces cerevisiae

EARLI1 is an Arabidopsis gene with pleiotropic effects previously shown to have auxiliary functions in protecting plants against freezing-induced cellular damage and promoting germinability under low-temperature and salinity stresses. Here we determined whether recombinant EARLI1 protein has anti-fungal activity. Recombinant EARLI1 protein lacking its signal peptide was produced in Escherichia coli BL21(DE3) using isopropyl β-d-1-thiogalactopyranoside (IPTG) induction and the prokaryotic expression vector pET28a. Expression of EARLI1 was analyzed by Western blotting and the protein was purified using affinity chromatography. Recombinant EARLI1 protein was applied to fungal cultures of Saccharomyces cerevisiae, Botrytis cinerea and Fusarium oxysporum, and membrane permeability was determined using SYTOX green. Full-length EARLI1 was expressed in S. cerevisiae from the GAL1 promoter using 2% galactose and yeast cell viability was compared to control cells. Our results indicated that application of recombinant EARLI1 protein to B. cinerea and F. oxysporum could inhibit the growth of the necrotrophic fungi. Besides, addition of the recombinant protein to liquid cultures of S. cerevisiae significantly suppressed yeast growth and cell viability by increasing membrane permeability, and in vivo expression of the secreted form of EARLI1 in S. cerevisiae also had a remarkable inhibition effect on the growth of yeast cells.

<italic>GhHyPRP4</italic>, a cotton gene encoding putative hybrid proline-rich protein, is preferentially expressed in leaves and involved in plant response to cold stress

Plant hybrid proline-rich proteins (HyPRPs) usually consist of an N-terminal signal peptide, a central proline-rich domain, and a conserved eight-cysteine motif C-terminal domain. In this study, one gene (designated as GhHyPRP4) encoding putative HyPRP was isolated from cotton cDNA library. Northern blot and quantitative reverse transcriptase-polymerase chain reaction analyses revealed that GhHyPRP4 was preferentially expressed in leaves. Under cold stress, GhHyPRP4 expression was significantly up-regulated in leaves of cotton seedlings. Using the genome walking approach, a promoter fragment of GhHyPRP4 gene was isolated from cotton genome. GUS (β-glucuronidase) gene driven by GhHyPRP4 promoter was specifically expressed in leaves and cotyledons of the transgenic Arabidopsis thaliana. Furthermore, GUS expression in leaves was remarkably induced by cold stress. Overexpression of GhHyPRP4 in yeast (Schizosaccharomyces pombe) significantly enhanced the cell survival rate upon treatment under -20°C for 60 h. These data suggested that GhHyPRP4 may be involved in plant response to cold stress during seedling development of cotton.

The HyPRP gene EARLI1 has an auxiliary role for germinability and early seedling development under low temperature and salt stress conditions in Arabidopsis thaliana

The effect of the hybrid proline-rich protein (HyPRP) gene EARLI1 on the rate of germination (germinability) of Arabidopsis seeds and seedling growth under low temperature and salt stress conditions was investigated. EARLI1 was induced during germination in embryonic tissues, and was strongly expressed in certain parts of young seedlings. Comparisons of control, overexpressing (OX), and knockout (KO) lines indicated that higher than wild type levels of EARLI1 improved germinability, root elongation, and reduction of sodium accumulation in leaves under salt stress, as well as germinability under low-temperature stress. Abscisic acid (ABA) contents were relatively low after prolonged salt stress, suggesting that EARLI1 has an ABA-independent effect on germinability under these conditions. Overexpression of EARLI1 during germination enhanced the sensitivity of seeds to exogenously applied ABA, suggesting that EARLI1 has an ABA-dependent negative effect on seed germinability under high ABA stress conditions. Well-known stress response marker genes such as COR15a, KIN1, P5SC1, and RD29 were unaffected whereas P5SC2, RD22, or RAB18 were only slightly affected in OX and KO plants. The pleiotropic effects of EARLI1 during stress and an absence of strong regulatory effects on stress marker genes suggest that this HyPRP gene has an auxiliary role for various stress protection responses in Arabidopsis.

Analysis of the hybrid proline-rich protein families from seven plant species suggests rapid diversification of their sequences and expression patterns.

BackgroundPlant hybrid proline-rich proteins (HyPRPs) are putative cell wall proteins consisting, usually, of a repetitive proline-rich (PR) N-terminal domain and a conserved eight-cysteine motif (8 CM) C-terminal domain. Understanding the evolutionary dynamics of HyPRPs might provide not only insight into their so far elusive function, but also a model for other large protein families in plants.ResultsWe have performed a phylogenetic analysis of HyPRPs from seven plant species, including representatives of gymnosperms and both monocot and dicot angiosperms. Every species studied possesses a large family of 14–52 HyPRPs. Angiosperm HyPRPs exhibit signs of recent major diversification involving, at least in Arabidopsis and rice, several independent tandem gene multiplications. A distinct subfamily of relatively well-conserved C-type HyPRPs, often with long hydrophobic PR domains, has been identified. In most of gymnosperm (pine) HyPRPs, diversity appears within the C-type group while angiosperms have only a few of well-conserved C-type representatives. Atypical (glycine-rich or extremely short) N-terminal domains apparently evolved independently in multiple lineages of the HyPRP family, possibly via inversion or loss of sequences encoding proline-rich domains. Expression profiles of potato and Arabidopsis HyPRP genes exhibit instances of both overlapping and complementary organ distribution. The diversified non-C-type HyPRP genes from recently amplified chromosomal clusters in Arabidopsis often share their specialized expression profiles. C-type genes have broader expression patterns in both species (potato and Arabidopsis), although orthologous genes exhibit some differences.ConclusionHyPRPs represent a dynamically evolving protein family apparently unique to seed plants. We suggest that ancestral HyPRPs with long proline-rich domains produced the current diversity through ongoing gene duplications accompanied by shortening, modification or loss of the proline-rich domains. Most of the diversity in gymnosperms and angiosperms originates from different branches of the HyPRP family. Rapid sequence diversification is consistent with only limited requirements for structure conservation and, together with high variability of gene expression patterns, limits the interpretation of any functional study focused on a single HyPRP gene or a couple of HYPRP genes in single plant species.

Cold responsive EARLI1 type HyPRPs improve freezing survival of yeast cells and form higher order complexes in plants

Plants have large families of proteins sharing a conserved eight-cysteine-motif (8CM) domain. The biological functions of these proteins are largely unknown. EARLI1 is a cold responsive Arabidopsis gene that encodes a hybrid proline-rich protein (HyPRP) with a three-domain architecture: a putative signal peptide at the N-terminus, a proline-rich domain (PRD) in the middle, and an 8CM domain at the C-terminus. We report here that yeast cells expressing different EARLI1 genes had significantly higher rates of freezing survival than empty-vector transformed controls. Arabidopsis plants with knocked down EARLI1 genes had an increased tendency for freezing-induced cellular damage. EARLI1-GFP fluorescence in transgenic plants and immunoblot analyses using protoplasts suggested cell wall localization for EARLI1 proteins. Immunoblot analyses showed that EARLI1 proteins form higher order complexes in plants, and that the PRD is a soluble and the 8CM an insoluble protein domain. We propose that EARLI1 proteins have a bimodular architecture in which the PRD may interact with the cell wall and the 8CM domain with the plasma membrane to protect the cells during freezing stress.

Developmental and Hormonal Regulation of Genes Coding for Proline-Rich Proteins in Female Inflorescences and Kernels of Maize

The pattern of expression of two genes coding for proteins rich in proline, HyPRP (hybrid proline-rich protein) and HRGP (hydroxyproline-rich glycoprotein), has been studied in maize (Zea mays) embryos by RNA analysis and in situ hybridization. mRNA accumulation is high during the first 20 d after pollination, and disappears in the maturation stages of embryogenesis. The two genes are also expressed during the development of the pistillate spikelet and during the first stages of embryo development in adjacent but different tissues. HyPRP mRNA accumulates mainly in the scutellum and HRGP mRNA mainly in the embryo axis and the suspensor. The two genes appear to be under the control of different regulatory pathways during embryogenesis. We show that HyPRP is repressed by abscisic acid and stress treatments, with the exception of cold treatment. In contrast, HRGP is affected positively by specific stress treatments.

A maize embryo-specific gene encodes a proline-rich and hydrophobic protein.

A gene from maize that encodes a hybrid proline-rich protein (HyPRP) formed by two well-defined domains, proline-rich and hydrophobic, respectively, has been characterized at the level of its structure and expression. The proline-rich domain is composed of elements PPYV and PPTPRPS, similar to those found in PRP proteins from soybean. The hydrophobic domain is rich in cysteine and is similar to seed proteins, mainly to a soybean hydrophobic seed protein. In maize, HyPRP is encoded by a single gene, and its mRNA accumulates in immature maize zygotic embryos, with a maximum accumulation between 12 and 18 days after pollination. The HyPRP mRNA can also be detected in ovary prior to pollination. In situ hybridization experiments on embryo sections show an expression of the gene in scutellum and in nonvascular cells from the embryo axis. Functional hypotheses related to HyPRP are discussed.