Pyr-miR171f-targeted <Emphasis Type="Italic">PyrSCL6</Emphasis> and <Emphasis Type="Italic">PyrSCL22</Emphasis> genes regulate shoot growth by responding to IAA signaling in pear

MicroRNA171 (miR171) is a highly conserved miRNA family, crucial for plant growth and development, and has been reported in Arabidopsis thaliana and tomato (Solanum lycopersicum), but the role of miR171 has not been explored in pear. In this study, an effort was made to decipher the mechanism underlying dwarf in ‘Zhongai 3’, of which the shoot length and shoot growth rate during the growing season were much less than those of the vigorous cultivar ‘Zaosu’, and the same for the indole-3-acetic acid (IAA) content in shoot tips after May 22, 2016. We identified a member of the miR171 family, which was most sensitive to IAA and targeted two genes conformed by 5′-RACE, and we named Pyr-miR171f. The two targets were named as PyrSCL6 and PyrSCL22, and contained a GRAS-conserved domain and encoded nucleus proteins. Quantitative RT-PCR analysis revealed that Pyr-miR171f was more abundant in ‘Zaosu’ shoot tips than in ‘Zhongai 3’ shoot tips, whereas the PyrSCL6 and PyrSCL22 mRNAs were more abundant in ‘Zhongai 3’ shoot tips than in ‘Zaosu’ shoot tips. The abundance of Pyr-miR171f and PyrSCL6 and PyrSCL22 mRNAs increased, but the trends were opposite between Pyr-miR171f and its target mRNAs in tissue culture seedlings treated by IAA. Our results suggest that IAA-induced miR171f negatively regulates the IAA signaling cascade via the GRAS pathway to maintain apical dominance. This work reveals a role for the miR171-SCL pathway in the dwarfing of ‘Zhongai 3’, and provides a theoretical basis for dwarf pear breeding.     

Determination S-Genotypes and Identification of Five Novel S-RNase Alleles in Wild Malus Species

Apple (Malus × domestica Borkh.) is a typical Rosaceae species that exhibits gametophytic self-incompatibility (GSI) controlled by polymorphic S-alleles. In this study, the S-alleles of wild Malus species were amplified, sequenced and compared using polymerase chain reaction (PCR) technology. 21 S-alleles were identified in 27 wild Malus species. The results indicated that the overwhelming majority of S-alleles between wild Malus species and cultivars shared identical sequences. Simultaneously, five new S alleles (designated S ₄₈ –S ₅₂ ) were identified in wild Malus species. There are the S ₄₈ -RNase in M. angustifolia (Ation) Michaux, S ₄₉ -RNase in M. orientalis Uglitzk. Ex Juz. and M. sylvestris (L.) Mill., S ₅₀ -RNase in M. tschonoskii (Maxim.) C.K. Schneid. and M. sieversii (Ldb.) Roem., S ₅₁ -RNase in M. komarovii (Sarg.) Rehd. and M. kansuensis (Batal.) C. K. Schneid., S ₅₂ -RNase in M. manshurica (Maxim.) V. Komorov wild Malus species. Additionally, an S ₁ -RNase was cloned in wild Malus prunifolia var. ringo, which have the same open reading frame as Malus × domestica cv. Fuji, but lacked whole intron.     

Screening and characterization of apple Rho-like GTPase (MdROPs) genes related to S-RNase mediated self-incompatibility

ROP/Rac GTPase is a conserved class of proteins which play diverse signalling roles in plants. They regulate many fundamental cellular processes such as F-actin dynamics, cell polarity and polar growth. Using apple genomic database analyses, nine ROP family members were cloned for the first time in a fruit tree (apple). Phylogenetic analyses indicated that the nine MdROPs were distributed into two groups, as previously described in the literature for model plants. Expression analyses show all MdROPs were highly expressed in pollen, in particular MdROP1, 3, 4 and 8. Yeast two hybrid and bimolecular fluorescence complementation analyses indicated MdROP8 interacts with S-RNase, a pistil determinate factor in gametophyte self-incompatibility. The pollen tube microtubule is shown to depolymerize in response to S-RNase treatment, during which the expression of MdROP8 rapidly decreased. These results indicate MdROP8 is related to S-RNase mediated self-incompatibility, and gives some useful evidence in modeling the relationship between cytoskeleton depolymerization and pollen tube growth inhibition during the apple SI reaction.     

An applicable nitrogen supply strategy for attached cultivation of Aucutodesmus obliquus

The “attached cultivation” method of microalgae in which the wet paste of algal biomass is attached onto supporting materials to form an immobilized biofilm layer, and the culture medium is supplied to this layer to provide nutrients and moisture for growth was highly efficient in biomass production and represents a promising technology to improve the biofuel industry. To optimize the nitrogen supply strategy for this attached cultivation method, the growth and total lipids accumulation properties for the green alga Aucutodesmus obliquus with this method were studied under different quantities of nitrogen source and different volumes of aqueous medium that continuously circulated inside the photobioreactor. Results showed that, compared with medium volume, the nitrogen quantity was a stronger factor affecting the growth and total lipid accumulation. An optimized nitrogen supply strategy for the attached cultivation of A. obliquus is proposed as circulating ca. 60 L of BG-11 medium containing 1/10 of nitrate concentration for 1 m² of cultivation surface. With this strategy, the attached A. obliquus accumulated biomass and total lipids simultaneously and obtained a high triacylglyceride productivity of 2.53 g m^?2 day^?1 in 7 days under subsaturated illumination of 100 μmol photons m^?2 s^?1. The water usage of 60 L m^?2 was potentially decreased to <2 L m^?2 if the nutrient supply was further improved. Dissolving the nitrogen source in small volume was the best way to efficiently utilize the nitrogen source with minimum of waste.     

Isolation and <Emphasis Type="Italic">S</Emphasis>-genotyping application of <Emphasis Type="Italic">S</Emphasis>-allelic polymorphic <Emphasis Type="Italic">MdSLFB</Emphasis>s in apple (<Emphasis Type="Italic">Malus domestica</Emphasis> Borkh.)

Apple (Malus domestica Borkh.) possesses gametophytic self-incompatibility (GSI) which is controlled by S-RNase in the pistil as well as a pollen S-determinant that has not been well characterized. The identification of S-locus F-box brother (SFBB) genes, which are good candidates for the pollen S-determinant in apple and pear, indicated the presence of multiple S-allelic polymorphic F-box genes at the S-locus. In apple, two SFBB gene groups have been described, while there are at least three groups in pear. In this report, we identified five MdSLFB (S-RNase-linked F-box) genes from four different S-genotypes of apple. These genes showed pollen- and S-allele-specific expression with a high polymorphism among S-alleles. The phylogenetic tree suggested that some of them belong to SFBBα or β groups as described previously, while others appear to be different from SFBBs. In particular, the presence of MdSLFB3 and MdSLFB9 suggested that there are more S-allelic polymorphic F-box gene groups in the S-locus besides α and β. Based on the sequence polymorphism of MdSLFBs, we developed an S-genotyping system for apple cultivars. In addition, we isolated twelve MdSLFB-like genes, which showed pollen-specific expression without S-allelic polymorphism.     

Characterization of three new S-alleles and development of an S-allele-specific PCR system for rapidly identifying the S-genotype in apple cultivars

Apple (Malus domestica Borkh), a member of the Rosaceae, shows gametophytic self-incompatibility (GSI) controlled by polymorphic S-alleles. Identifying the S-genotypes of apple cultivars can be applied on correct assignment of apple cultivars to cross-compatibility groups, which is important for the efficient production of apple fruit. This study characterized three new S-alleles (designated S ₄₄ , S ₄₅ , and S ₄₆ ) in apple and developed an efficient analysis method that can be used to characterize S-genotypes by utilizing allele-specific polymerase chain reaction rapidly. Nineteen allele-specific primers were selectively designed to identify different alleles. Using this method, S-genotypes of 157 apple cultivars were identified.     

Stomatal movement in response to long distance-communicated signals initiated by heat shock in partial roots of Commelina communis L.

The systematic or long-distance signal transmission plays crucial roles in animal lives. Compared with animals, however, much less is known about the roles of long-distance signal communication in plant lives. Using the model plant Commelina communis L., we have probed the root to shoot communication mediated by heat-shock signals. The results showed that a heat shock of 5 min at 40℃ in partial roots, i.e. half or even 1/4 root system, could lead to a significant decrease in stomatal conductance. The regulation capability depends on both heat shock temperature and the amount of root system, i.e. with higher temperature and more roots stressed, the leaf conductance would decrease more significantly. Interestingly, the stomatal regulation by heat shock signal is in a manner of oscillation: when stomata conductance decreased to the lowest level within about 30 min, it would increase rapidly and sometimes even exceed the initial level, and after several cycles the stomata conductance would be finally stabilized at a lower level. Feeding xylem sap collected from heat-shocked plants could lead to a decrease in stomata conductance, suggesting that the heat shock-initiated signal is basically a positive signal. Further studies showed that heat shock was not able to affect ABA content in xylem sap, and also, not able to lead to a decrease in leaf water status, which suggested that the stomatal regulation was neither mediated by ABA nor by a hydraulic signal. Heat shock could lead to an increase in xylem sap H₂O₂ content, and moreover, the removal of H₂O₂ by catalase could partially recover the stomatal inhibition by xylem sap collected from heat-shocked plants, suggesting that H₂O₂ might be able to act as one of the root signals to control the stomatal movement. Due to the fact that heat-shock and drought are usually two concomitant stresses, the stomatal regulation by heat-shock signal should be of significance for plant response to stresses. The observation for the stomatal regulation in an oscillation manner by presently identified new signals should contribute to further understanding of the mystery for the pant systematic signaling in response to stresses.     

城市生活废水用于产油微藻培养

将废水与产油微藻培养结合起来,可以实现废水的无害化处理,还可为微藻的培养提供营养组分和大量水源。利用高产油栅藻,以城市生活废水为水源,在气泡柱式光反应器中,考察了添加不同营养组分对栅藻细胞的生长、生物质产量、总脂含量以及氮磷的去除情况的影响。结果表明：生活废水非常适合于产油微藻的培养,利用生活废水进行微藻培养中,仅需补充添加无机氮、无机磷、柠檬酸铁铵以及微量元素。但这些营养组分的加入量对藻细胞的生长、生物量和油脂积累有重要影响。在优化的废水培养基中微藻细胞浓度可达8.0 g/L左右,远高于标准BG11培养基5.0 g/L的水平。微藻细胞对于无机氮与磷有着高的吸收能力,在废水中加入185.25 mg/L以下无机氮,16.1 mg/L以下无机磷的条件下培养3~4 d后,培养液水体中未检测到有氮磷残留。由此表明利用城市生活废水培养含油微藻可以在获得微藻油脂产品的同时实现水体的无害化处理。     

Ubiquitination of S4-RNase by S-LOCUS F-BOX LIKE2 Contributes to Self-Compatibility of Sweet Cherry ‘Lapins’

Recent studies have shown that loss of pollen-S function in S₄′ pollen from sweet cherry (Prunus avium) is associated with a mutation in an S haplotype-specific F-box4 (SFB4) gene. However, how this mutation leads to self-compatibility is unclear. Here, we examined this mechanism by analyzing several self-compatible sweet cherry varieties. We determined that mutated SFB4 (SFB4ʹ) in S4′ pollen (pollen harboring the SFB4ʹ gene) is approximately 6 kD shorter than wild-type SFB4 due to a premature termination caused by a four-nucleotide deletion. SFB4′ did not interact with S-RNase. However, a protein in S4′ pollen ubiquitinated S-RNase, resulting in its degradation via the 26S proteasome pathway, indicating that factors in S4′ pollen other than SFB4 participate in S-RNase recognition and degradation. To identify these factors, we used S₄-RNase as a bait to screen S4′ pollen proteins. Our screen identified the protein encoded by S₄-SLFL2, a low-polymorphic gene that is closely linked to the S-locus. Further investigations indicate that SLFL2 ubiquitinates S-RNase, leading to its degradation. Subcellular localization analysis showed that SFB4 is primarily localized to the pollen tube tip, whereas SLFL2 is not. When S₄-SLFL2 expression was suppressed by antisense oligonucleotide treatment in wild-type pollen tubes, pollen still had the capacity to ubiquitinate S-RNase; however, this ubiquitin-labeled S-RNase was not degraded via the 26S proteasome pathway, suggesting that SFB4 does not participate in the degradation of S-RNase. When SFB4 loses its function, S₄-SLFL2 might mediate the ubiquitination and degradation of S-RNase, which is consistent with the self-compatibility of S4′ pollen.

In sweet cherry (Prunus avium), self-incompatibility is mainly controlled by the S-locus, which is located at the end of chromosome 6 (Akagi et al., 2016; Shirasawa et al., 2017). Although the vast majority of sweet cherry varieties show self-incompatibility, some self-compatible varieties have been identified, most of which resulted from the use of x-ray mutagenesis and continuous cross-breeding (Ushijima et al., 2004; Sonneveld et al., 2005). At present, naturally occurring self-compatible varieties are rare (Marchese et al., 2007; Wünsch et al., 2010; Ono et al., 2018). X-ray-induced mutations that have given rise to self-compatibility include a 4-bp deletion (TTAT) in the gene encoding an SFB4′ (S-locus F-box 4′) protein, located in the S-locus and regarded as the dominant pollen factor in self-incompatibility. This mutation is present in the first identified self-compatible sweet cherry variety, ‘Stellar’, as well as in a series of its self-compatible descendants, including ‘Lapins’, ‘Yanyang’, and ‘Sweet heart’ (Lapins, 1971; Ushijima et al., 2004). Deletion of SFB3 and a large fragment insertion in SFB5 have also been identified in other self-compatible sweet cherry varieties (Sonneveld et al., 2005; Marchese et al., 2007). Additionally, a mutation not linked to the S-locus (linked instead to the M-locus) could also cause self-compatibility in sweet cherry and closely related species such as apricot (Prunus armeniaca; Wünsch et al., 2010; Zuriaga et al., 2013; Muñoz-Sanz et al., 2017; Ono et al., 2018). Much of the self-compatibility in Prunus species seems to be closely linked to mutation of SFB in the S-locus (Zhu et al., 2004; Muñoz-Espinoza et al., 2017); however, the mechanism of how this mutation of SFB causes self-compatibility is unknown.The gene composition of the S-locus in sweet cherry differs from that of other gametophytic self-incompatible species, such as apple (Malus domestica), pear (Pyrus spp.), and petunia (Petunia spp.). In sweet cherry, in addition to a single S-RNase gene, the S-locus contains one SFB gene, which has a high level of allelic polymorphism, and three SLFL (S-locus F-box-like) genes with low levels of, or no, allelic polymorphism (Ushijima et al., 2004; Matsumoto et al., 2008). By contrast, the apple, pear, and petunia S-locus usually contains one S-RNase and 16 to 20 F-box genes (Kakui et al., 2011; Okada et al., 2011, 2013; Minamikawa et al., 2014; Williams et al., 2014a; Yuan et al., 2014; Kubo et al., 2015; Pratas et al., 2018). The F-box gene, named SFBB (S-locus F-box brother) in apple and pear and SLF (S-locus F-box) in petunia, exhibits higher sequence similarity with SLFL than with SFB from sweet cherry (Matsumoto et al., 2008; Tao and Iezzoni, 2010). The protein encoded by SLF in the petunia S-locus is thought to be part of an SCF (Skp, Cullin, F-box)-containing complex that recognizes nonself S-RNase and degrades it through the ubiquitin pathway (Kubo et al., 2010; Zhao et al., 2010; Chen et al., 2012; Entani et al., 2014; Li et al., 2014, 2016, 2017; Sun et al., 2018). In sweet cherry, a number of reports have described the expression and protein interactions of SFB, SLFL, Skp1, and Cullin (Ushijima et al., 2004; Matsumoto et al., 2012); however, only a few reports have examined the relationship between SFB/SLFL and S-RNase (Matsumoto and Tao, 2016, 2019), and none has investigated whether the SFB/SLFL proteins participate in the ubiquitin labeling of S-RNase.Although the function of SFB4 and SLFL in self-compatibility is unknown, the observation that S4′ pollen tubes grow in sweet cherry pistils that harbor the same S alleles led us to speculate that S4′ pollen might inhibit the toxicity of self S-RNase. In petunia, the results of several studies have suggested that pollen tubes inhibit self S-RNase when an SLF gene from another S-locus haplotype is expressed (Sijacic et al., 2004; Kubo et al., 2010; Williams et al., 2014b; Sun et al., 2018). For example, when SLF2 from the S₇ haplotype is heterologously expressed in pollen harboring the S₉ or S₁₁ haplotype, the S₉ or S₁₁ pollen acquire the capacity to inhibit self S-RNase and break down self-incompatibility (Kubo et al., 2010). The SLF2 protein in petunia has been proposed to ubiquitinate S₉-RNase and S₁₁-RNase and lead to its degradation through the 26S proteasome pathway (Entani et al., 2014). If SFB/SLFL in sweet cherry have a similar function, the S4′ pollen would not be expected to inhibit self S₄-RNase, prompting the suggestion that the functions of SFB/SLFL in sweet cherry and SLF in petunia vary (Tao and Iezzoni, 2010; Matsumoto et al., 2012).In this study, we used sweet cherry to investigate how S4′ pollen inhibits S-RNase and causes self-compatibility, focusing on the question of whether the SFB/SLFL protein can ubiquitinate S-RNase, resulting in its degradation.