Characterization and Potential Action Mode Divergences of Homologous ACO1 Genes during the Organ Development and Ripening Process between Non-Climacteric Grape and Climacteric Peach.

Ethylene is one crucial phytohormone modulating plants' organ development and ripening process, especially in fruits, but its action modes and discrepancies in non-climacteric grape and climacteric peach in these processes remain elusive. This work is focused on the action mode divergences of ethylene during the modulation of the organ development and ripening process in climacteric/non-climacteric plants. We characterized the key enzyme genes in the ethylene synthesis pathway, VvACO1 and PpACO1, and uncovered that their sequence structures are highly conserved, although their promoters exhibit important divergences in the numbers and types of the cis-elements responsive to hormones, implying various responses to hormone signals. Subsequently, we found the two have similar expression modes in vegetative organ development but inverse patterns in reproductive ones, especially in fruits. Then, VvACO1 and PpACO1 were further validated in promoting fruit ripening functions through their transient over-expression/RNAi-expression in tomatoes, of which the former possesses a weaker role than the latter in the fruit ripening process. Our findings illuminated the divergence in the action patterns and function traits of the key VvACO1/PpACO1 genes in the tissue development of climacteric/non-climacteric plants, and they have implications for further gaining insight into the interaction mechanism of ethylene signaling during the modulation of the organ development and ripening process in climacteric/non-climacteric plants.

Current Situation of Fire Blight in China.

Fire blight, caused by the plant-pathogenic bacterium Erwinia amylovora, is a devastating disease that occurs on rosaceous plants, including pears and apples. E. amylovora is indigenous to North America and was spread to the Eurasian continent in the second half of the 20th century through contaminated plant materials. In 2016, fire blight was first observed in Yili, Xinjiang Province, in Northwestern China. Since then, it has spread to most pear-producing regions in Xinjiang Province and parts of Gansu Province. The disease has caused severe damage to China's pear and apple industries, including the 2017 disease epidemic in Korla, Xinjiang, which caused an overall yield reduction of 30 to about 50% in Korla and the destruction of over 1 million pear trees. Over the past few years, a combined effort of research, extension, and education by the Chinese government, scientists, and fruit growers has greatly alleviated outbreaks and epidemics in affected regions while successfully limiting the further spread of fire blight to new geographical regions. Here, we review the occurrence, spread, and damage of this disease to the Chinese fruit industry, as well as the management options used in China and their outcomes. We also discuss future perspectives for restraining the spread and alleviating the damage of fire blight in China.

Two ways to die: Species dependent PCD modes in grapevine cells.

Programmed cell death (PCD) is considered as a hallmark of strain-specific immunity. In contrast, generic basal immunity is thought to act without PCD. This classical bifurcation has been questioned during recent years. Likewise, the role of jasmonate signalling for these two modes of innate immunity has remained ambiguous. We have addressed both questions using two closely related grapevine cell lines (V. rupestris, V. vinifera cv. 'Pinot Noir') that contrast in their cell-death response to the bacterial elicitor harpin and the hormonal trigger methyl jasmonate (MeJA). We follow different cellular (loss of membrane integrity, mortality), molecular (induction of transcripts for phytoalexin synthesis and for metacaspases), as well as metabolic (sphingolipid profiles) responses to the two triggers in the two cell lines. The role of NADPH oxidases and induction of transcripts for the class-II metacaspases MC5 differ qualitatively between the two cell lines. We tested a possible role of sphingolipid metabolism but can rule this out. We propose a model, where V. rupestris, originating from co-evolution with several biotrophic pathogens, readily activates a hypersensitive cell death in response to harpin, while the context of MeJA-induced cell death in 'Pinot Noir' might not be related to immunity at all. We propose that the underlying signalling is modular, recruiting metacaspases differently depending on upstream signalling.

Assessment of 'Cabernet Sauvignon' Grape Quality Half-Véraison to Maturity for Grapevines Grown in Different Regions.

Grapes are widely cultivated around the world and their quality has distinct regional characteristics. In this study, the qualitative characteristics of the 'Cabernet Sauvignon' grape variety in seven regions, from half-véraison to maturity, were analyzed comprehensively at physiological and transcriptional levels. The results indicated that the quality traits of 'Cabernet Sauvignon' grapes in different regions were significantly different with obvious regionality. Total phenols, anthocyanins, and titratable acids were the main factors of the regionality of berry quality, which were very sensitive to changes in the environment. It should be noted that the changes in titrating acids and total anthocyanin of berries vary greatly from half-véraison to maturity between regions. Moreover, the transcriptional analysis showed that the co-expressed genes between regions characterized the core transcriptome of berry development, while the unique genes of each region reflected the regionality of berries. The differentially expressed genes (DEGs) between half-véraison and maturity can be used to demonstrate that the environment of the regions could promote or inhibit gene expression. The functional enrichment suggested that these DEGs help to understand the interpretation of the plasticity of the quality composition of grapes according to the environment. Taken together, the information generated by this study could contribute to the development of viticultural practices aimed at making better use of native varieties for the development of wines with regional characteristics.

Identification and Characterization of AUXIN Response Factor Gene Family Reveals Their Regulatory Network to Respond the Multi-Hormones Crosstalk during GA-Induced Grape Parthenocarpic Berry.

Exogenous gibberellin (GA) was widely used to improve berry quality through inducing parthenocarpic seedless berries in grapes. We revealed that auxin response factors (ARFs), the key transcription factors in response to auxin, might respond to GA involving modulation of grape parthenocarpy. However, the underlying molecular mechanism in this process remains yet unclear. Here, a total of 19 VvARF members were identified in the ovaries during GA-induced grapes' parthenocarpy. Interestingly, almost all members were GA-responsive factors, of which 9 could be classified in plant hormone signal transduction (KO04075) and involved in the tryptophan metabolic pathway (K14486). Moreover, VvARFs were predicted to have 310 interacted proteins involved in 19 KEGG pathways. Of them, 32 interacted proteins participated in the KO04075 pathway, including auxin (IAA), salicylic acid (SA), abscisic acid (ABA), cytokinin (CTK), and ethylene signaling pathways by responding to GA-mediated multi-hormone crosstalk. Further analysis demonstrated that VvARF4-2 might be the major factor in the modulation of GA-induced parthenocarpy via the crosstalk of IAA, CTK, SA, and ethylene signaling, followed by VvARF6-1 and VvARF9 involved in SA and ABA signaling pathways, respectively. Finally, we developed a VvARFs-mediated regulatory network by responding to GA-mediated multi-hormone crosstalk during grape parthenocarpy. Collectively, our findings provided novel insights into the regulatory network of VvARFs in GA-guided multi-hormone signaling to modulate grape parthenocarpy, which has great implications for the molecular breeding of high quality seedless grape berries.

Transcriptional Profiling of Resistant and Susceptible Cultivars of Grapevine (Vitis L.) Reveals Hypersensitive Responses to Plasmopara viticola.

Grapevine downy mildew is the most serious disease of grapevine cultivars that affects the rate of resistance/susceptibility to Plasmopara viticola. In this study, we used the susceptible cultivar "Zitian Seedless" and the resistant cultivar "Kober 5BB" as materials to determine the transcriptome differences and phenotypes of the leaves after inoculation with downy mildew. The differences in microstructures and molecular levels were compared and analyzed. Fluorescence staining and microscopic observations confirmed that hypersensitive cell death occurred around the stomata in "Kober 5BB" infected by downy mildew zoospores. Meanwhile, transcriptomic profiling indicated that there were 11,713 and 6,997 gene expression differences between the resistant and susceptible cultivars at 72 h after inoculation when compared to control (0 h), respectively. The differentially expressed genes of the two cultivars are significantly enriched in different pathways, including response to plant-pathogen interaction, mitogen-activated protein kinase (MAPK) signaling pathway, plant hormone signal transduction, phenylpropanoid, and flavonoid biosynthesis. Furthermore, the results of functional enrichment analysis showed that H2O2 metabolism, cell death, reactive oxygen response, and carbohydrate metabolism are also involved in the defense response of "Kober 5BB," wherein a total of 322 key genes have been identified. The protein interaction network showed that metacaspases (MCAs), vacuolar processing enzymes (VPEs), and Papain-like cysteine proteases (PLCPs) play an important role in the execution of hypersensitive responses (HR). In conclusion, we demonstrated that HR cell death is the key strategy in the process of grape defense against downy mildew, which may be mediated or activated by Caspase-like proteases.

"The PLCP gene family of grapevine (Vitis vinifera L.): characterization and differential expression in response to Plasmopara Viticola".

BACKGROUND: Papain-like cysteine proteases (PLCPs), a large group of cysteine proteases, are structurally related to papain. The members belonging to PLCPs family contribute to plant immunity, senescence, and defense responses in plants. The PLCP gene family has been identified in Arabidopsis, rice, soybean, and cotton. However, no systematic analysis of PLCP genes has been undertaken in grapevine. Since Plasmopara viticola as a destructive pathogen could affect immunity of grapes in the field, we considered that the members belonged to PLCPs family could play a crucial role in defensive mechanisms or programmed cell death. We aimed to evaluate the role of PLCPs in 2 different varieties of grapevines and compared the changes of their expressions with the transcriptional data in response to P. viticola. RESULTS: In this study, 23 grapevine PLCP (VvPLCP) genes were identified by comprehensive bioinformatics analysis. Subsequently, the chromosomal localizations, gene structure, conserved domains, phylogenetic relationship, gene duplication, and cis-acting elements were analyzed. Numerous cis-acting elements related to plant development, hormone, and stress responses were identified in the promoter of the VvPLCP genes. Phylogenetic analysis grouped the VvPLCP genes into nine subgroups. The transcription of VvPLCP in different inoculation time points and varieties indicated that VvPLCP may have vital functions in grapevine defense against Plasmopara viticola. According to transcriptome data and qPCR analysis, we observed the increasing expression levels of VvRD21-1 at 72 h after inoculation in resistant variety, inferring that it was related to grape downy mildew resistance. Meanwhile, 3 genes including VvXBCP1, VvSAG12-1, and VvALP1 showed higher expression at 24 h after pathogen inoculation in the susceptible variety and might be related to the downy mildew phenotype. We nominated these four genes to function during hypersensitive response (HR) process, inferring that these genes could be associated with downy mildew resistance in grapes. CONCLUSIONS: Our results provide the reference for functional studies of PLCP gene family, and highlight its functions in grapevine defense against P. viticola. The results help us to better understand the complexity of the PLCP gene family in plant immunity and provide valuable information for future functional characterization of specific genes in grapevine.

Review: Microtubules monitor calcium and reactive oxygen species signatures in signal transduction.

Signal transductions require calcium (Ca2+) or reactive oxygen species (ROS) signatures, which act as chemical and electrical signals in response to various biotic and abiotic stresses. Calcium as an ion or second messenger affects the membrane potential and microtubules (MTs) dynamicity, while MTs can modulate auto-propagating waves of calcium and ROS signatures in collaboration with ion channels depending on the stimulus type. Thus, in the current review, we highlight advances in research focused on the relationship between dynamic MTs and calcium and ROS signatures in short-distance transmission. The challenges of Ca2+-MTs-ROS crosstalk in cold sensing are addressed, which could suggest the prioritization of ROS or Ca2+ in signalling.

Comparative transcriptome analysis provides insight into regulation pathways and temporal and spatial expression characteristics of grapevine (Vitis vinifera) dormant buds in different nodes.

BACKGROUND: Bud dormancy is a strategic mechanism plants developed as an adaptation to unfavorable environments. The grapevine (Vitis vinifera) is one of the most ancient fruit vine species and vines are planted all over the world due to their great economic benefits. To better understand the molecular mechanisms underlying bud dormancy between adjacent months, the transcriptomes of 'Rosario Bianco' grape buds of 6 months and three nodes were analyzed using RNA-sequencing technology and pair-wise comparison. From November to April of the following year, pairwise comparisons were conducted between adjacent months. RESULTS: A total of 11,647 differentially expressed genes (DEGs) were obtained from five comparisons. According to the results of cluster analysis of the DEG profiles and the climatic status of the sampling period, the 6 months were divided into three key processes (November to January, January to March, and March to April). Pair-wise comparisons of DEG profiles of adjacent months and three main dormancy processes showed that the whole grapevine bud dormancy period was mainly regulated by the antioxidant system, secondary metabolism, cell cycle and division, cell wall metabolism, and carbohydrates metabolism. Additionally, several DEGs, such as VvGA2OX6 and VvSS3, showed temporally and spatially differential expression patterns, which normalized to a similar trend during or before April. CONCLUSION: Considering these results, the molecular mechanisms underlying bud dormancy in the grapevine can be hypothesized, which lays the foundation for further research.

Molecular cloning and functional characterization of a seed-specific VvßVPE gene promoter from Vitis vinifera.

MAIN CONCLUSION: The grapevine VvßVPE promoter is specifically expressed in the seed. The - 1306~- 1045 bp core region restricts expression in other tissues and organs. Vacuolar processing enzyme (VPE) is a cysteine proteinase regulating vacuolar protein maturation and executing programmed cell death (PCD) in plants. Vitis vinifera (Vv)ßVPE is a ß-type VPE showing seed-specific expression that processes seed proteins during ovule development. However, the regulation of the seed-specific gene expression is far from understood. In this study, we characterize VvßVPE promoter (pVvßVPE) from 12 seeded and seedless grape genotypes. 94.56% of the pVvßVPE coding sequence is consistent. Two ßVPE promoters were constructed and transformed into Arabidopsis thaliana via ß-glucuronidase (GUS) fused expression vectors, using cv. Pinot Noir and cv. Thompson as seed and seedless candidates. GUS staining in different tissues and organs revealed that VvßVPE expresses specifically in the embryo, including the cotyledon, hypocotyl and suspensor, but not in the leaf, stem, root or flowers of the seedling. Using promoter deletion analysis, we created four incomplete VvßVPE promoters and found each pVvßVPE deletion could drive GUS gene to express in seeds. Interestingly, seed specificity disappeared when the promoter missed the core - 1306~- 1045 bp region. All deletion promoters presenting various quantified GUS activities indicate that the region - 1704~- 1306 bp inhibits, and the region - 705~- 861 bp promotes gene expression of VvßVPE. Our results demonstrate that pVvßVPE is a seed-specific promoter in both seeded and seedless grapes. Moreover, the core region of pVvßVPE (- 1306~- 1045 bp) is the key one responsible for seed-specific expression.

Two grapevine metacaspase genes mediate ETI-like cell death in grapevine defence against infection of Plasmopara viticola.

Metacaspase, as hypersensitive response (HR) executors, has been identified in many plant species. Previously, the entire gene family of metacaspase has been uncovered, but there are still questions that remain unclear regarding HR-regulating gene members. In this study, based on metacaspase expression during different grapevine genotypes interacting with Plasmopara viticola, we identified MC2 and MC5 as candidates involved in HR. We overexpressed both metacaspases as GFP fusions in tobacco BY-2 cells to address subcellular localization and cellular functions. We found MC2 located at the ER, while MC5 was nucleocytoplasmic. In these overexpressor lines, cell death elicited by the bacterial protein harpin, is significantly enhanced, indicating MC2 and MC5 mediated defence-related programmed cell death (PCD). This effect was mitigated, when the membrane-located NADPH oxidase was inhibited by the specific inhibitor diphenylene iodonium, or when cells were complemented with methyl jasmonate, a crucial signal of basal immunity. Both findings are consistent with a role of MC2 and MC5 in cell death-related immunity. Using a dual-luciferase reporter system in grapevine cells we demonstrated both MC2 and MC5 promoter alleles from V. rupestris were more responsive to harpin than those from V. vinifera cv 'Müller-Thurgau', while they were not induced by MeJA as signal linked with basal immunity. These findings support a model, where MC2 and MC5 act specifically as executors of the HR.

Vacuolar processing enzyme (VvßVPE) from Vitis vinifera, processes seed proteins during ovule development, and accelerates seed germination in VvßVPE heterologously over-expressed Arabidopsis.

Vacuolar processing enzymes (VPEs), belonging to cysteine protease, are responsible for processing seed protein during maturation. Stenospermocarpic grapes occur self-abortion in fertilized embryos during the ovule development, which affects the formation of matured seed proteins. However, little is known about VPE functions in ovule self-defeating. Here, we investigated the role of one seed-type VPE gene, VvßVPE. Sequence analysis showed that all ORFs (Open reading frames) of VvßVPE from 19 seed/seedless genotypes are highly conserved. At the transcriptional level, VvßVPE was specifically expressed during ovule development, with distinct expression patterns: it increased gradually in seeded grapes; while weakly expressed in seedless grapes. Whereas, at the translational level, 3 forms of VvßVPE were expressed during ovule development in seeded grape: precursor ßVPE (pßVPE), intermediate ßVPE (ißVPE) and finally, active mature ßVPE (mßVPE). By contrast, in seedless grape, VvßVPE only exists as pßVPE at whole developmental stage of ovule. for confirming these expression patterns, 12 seeded/seedless genotypes were sampled and analyzed. Furthermore, VPE enzyme activity was increased in Arabidopsis overexpressing VvßVPE, leading to faster germination. Our study indicated that VvßVPE is essential for grapevine ovule maturation through various forms and is involved in seed germination.

Gene Cloning, Expression and Enzyme Activity of Vitis vinifera Vacuolar Processing Enzymes (VvVPEs).

Vacuolar processing enzymes (VPEs) have received considerable attention due to their caspase-1-like activity and ability to regulate programmed cell death (PCD), which plays an essential role in the development of stenospermocarpic seedless grapes ovules. To characterize VPEs and the relationship between stenospermocarpic grapes and the VPE gene family, we identified 3 Vitis vinifera VPE genes (VvßVPE, VvγVPE, and VvδVPE) from the PN40024 grape genome and cloned the full-length complementary DNAs (cDNAs) from the 'Vitis vinifera cv. Pinot Noir' and 'Vitis vinifera cv. Thompson Seedless' varietals. Each of the VPEs contained a typical catalytic dyad [His (177), Cys (219)] and substrate binding pocket [Arg (112), Arg (389), Ser (395)], except that Ser (395) in the VvγVPE protein sequence was replaced with alanine. Phylogenetic analysis of 4 Arabidopsis thaliana and 6 Vitis vinifera VPEs revealed that the 10 VPEs form 3 major branches. Furthermore, the 6 grapevine VPEs share a similar gene structure, with 9 exons and 8 introns. The 6 grapevine VPEs are located on 3 different chromosomes. We also tested the enzymatic activity of recombinant VPEs expressed in the Pichia Pastoris expression system and found that the VvVPEs exhibit cysteine peptidase activity. Tissue-specific expression analysis showed that VvδVPE is only expressed in flowers, buds and ovules, that VvγVPE is expressed in various tissues, and that VvßVPE was expressed in roots, flowers, buds and ovules. The results of quantitative real-time PCR (qRT-PCR) suggested that VvßVPE in seeded grapes increased significantly at 30 days after full-bloom (DAF), close to the timing of endosperm abortion at 32 DAF. These results suggested that VvßVPE is related to ovule abortion in seedless grapes. Our experiments provide a new perspective for understanding the mechanism of stenospermocarpic seedlessness and represent a useful reference for the further study of VPEs.

The metacaspase gene family of Vitis vinifera L.: characterization and differential expression during ovule abortion in stenospermocarpic seedless grapes.

In both plants and animals, programmed cell death (PCD) is an indispensable process that removes redundant cells. In seedless grapes (Vitis vinifera), abnormal PCD in ovule cells and subsequent ovule abortion play key roles in stenospermocarpy. Metacaspase, a type of cysteine-dependent protease, plays an essential role in PCD. To reveal the characteristics of the metacaspase (MC) gene family and the relationship between metacaspases and the seedless trait, we identified the 6 V. vinifera metacaspases VvMC1-VvMC6, from the grape genome, using BLASTN against the 9 known Arabidopsis metacaspases. We also obtained full-length cDNAs by RT-PCR. Each of the 6 grape metacaspases contains small (p10-like) and a large (p20-like) conserved structural domains. Phylogenetic analysis of 6 grape and 9 Arabidopsis metacaspases showed that all metacaspases could be grouped into two classes: Type I and Type II. Each phylogenetic branch shares a similar exon/intron structure. Furthermore, the putative promoters of the grape metacaspases contained cis-elements that are involved in grape endosperm development. Moreover, expression analysis of metacaspases using real-time quantitative PCR demonstrated that VvMC1 and VvMC2 were able to be detected in any tissue, and VvMC3, VvMC4, VvMC5 and VvMC6 exhibited tissue-specific expression. Lastly, in cv. Thompson seedless grapes VvMC1, VvMC3, and VvMC4 were significantly up-regulated at the 35 DAF during ovule development, roughly same stage as endosperm abortion. In addition, the expression trend of VvMC2 and VvMC5 was similar between cv. Pinot Noir and cv. Thompson grape ovule development and that of VvMC6 was sustained in a relatively low level except the expression of cv. Pinot Noir significantly up-regulated in 25 DAF. Our data provided new insights into PCD by identifying the grape metacaspase gene family and provide a useful reference for further functional analysis of metacaspases in grape.