The White Clover TrMYB33-TrSAMS1 Module Contributes to Drought Tolerance by Modulation of Spermidine Biosynthesis via an ABA-Dependent Pathway.

Spermidine is well known to accumulate in plants exposed to drought, but the regulatory network associated with its biosynthesis and accumulation and the underlying molecular mechanisms remain unclear. Here, we demonstrated that the Trifolium repens TrMYB33 relayed the ABA signal to modulate drought-induced spermidine production by directly regulating the expression of TrSAMS1, which encodes an S-adenosylmethionine synthase. This gene was identified by transcriptome and expression analysis in T. repens. TrSAMS1 overexpression and its pTRV-VIGS-mediated silencing demonstrated that TrSAMS1 is a positive regulator of spermidine synthesis and drought tolerance. TrMYB33 was identified as an interacting candidate through yeast one-hybrid library screening with the TrSAMS1 promoter region as the bait. TrMYB33 was confirmed to bind directly to the predicted TAACCACTAACCA (the TAACCA MYB binding site is repeated twice in tandem) within the TrSAMS1 promoter and to act as a transcriptional activator. Additionally, TrMYB33 contributed to drought tolerance by regulating TrSAMS1 expression and modulating spermidine synthesis. Additionally, we found that spermidine accumulation under drought stress depended on ABA and that TrMYB33 coordinated ABA-mediated upregulation of TrSAMS1 and spermidine accumulation. This study elucidated the role of a T. repens MYB33 homolog in modulating spermidine biosynthesis. The further exploitation and functional characterization of the TrMYB33-TrSAMS1 regulatory module can enhance our understanding of the molecular mechanisms responsible for spermidine accumulation during drought stress.

First report of leaf spot caused by Colletotrichum siamense on Michelia alba in China.

Michelia alba (common name: white champaca), native to Indonesia, is a preciously ornamental and medicinal plant in the west and southeast of China and widely distributed in Nanning, Guangxi, China (Hou et al. 2018). In May 2020, a foliar disease of M. alba was observed in Nanning (22°51' N; 108°17' E), Guangxi, China, present on ca. 20-30% of the leaves. The disease began to develop from the margins of leaves in most cases. The symptoms recorded were light yellow spots, which gradually developed into ellipsoidal to irregular brown spots, surrounded by a wide yellow halo. The spots gradually enlarged in size and became grey-brown, with the dimension of 3.5 × 2.8 to 11.0 × 3.5 cm, even more than half of leaf area. In the later stage of infection, these spots coalesced resulting in necrosis and early shedding of the leaves. Sometimes black acervuli were observed on some lesions. For isolation of the fungus, ten symptomatic leaves were randomly sampled from five trees and washed with sterile water. Small pieces of infected tissue (about 4 mm2) were surface disinfected in 75% alcohol for 30 s and in 0.1% aqueous solution of mercury chloride for 1 min. Finally these tissue pieces were rinsed three times with sterile water, plated on potato dextrose agar (PDA) and then incubated for 7 days at 28℃ with a photoperiod of 12 h. Fifteen strains with similar morphological characterizations were isolated, and five representative isolates (BL-1 to BL-5) were purified. These cultures gave rise to grey-white colonies with bright orange conidial masses with contained one-celled, hyaline, guttulate conidia, measuring 12.68-20.70 × 4.27-7.84 µm (average 15.36 × 5.35 µm, n=100). Appressoria formed from conidia were brown, ellipsoidal or inverted trapezoid and measured 6.36-12.13 × 5.07-7.39 µm (average 8.29 × 6.36 µm, n=30). These morphological characteristics were similar to those of the Colletotrichum gloeosporioides species complex (Weir et al. 2012). To confirm identification, genomic DNA from mycelium of these five isolates was extracted, and the sequence of internal transcribed spacer (ITS), chitin synthase (CHS-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), calmodulin (CAL) and ß-tubulin (TUB2) were amplified (Zhang et al. 2020), and the GenBank accession numbers for the sequences were MW186173 to MW186177 (ITS), MW161290 to 161294 (CHS-1), MW161295 to MW161299 (GAPDH), MW161285 to 161289 (ACT), MW084710 to 084714 (CAL) and MW161300 to MW161304 (TUB2). The phylogenetic tree of six combined genes of the five isolates clustered with Colletotrichum siamense strains (CBS 125378, ICMP 17795 and ICMP 18121). Therefore, the isolates were identified as C. siamense. Five isolates (BL-1 to BL-5) were tested for pathogenicity. Wounded and unwounded detached healthy leaves were inoculated using mycelial discs (5 mm in diameter) and conidial suspensions (with the concentration of 1 × 105 conidia/ml) at the same time, incubated in a growth chamber at 25-30℃ (85-90% relative humidity, with a photoperiod of 12 h). Three leaves (wounded left half blade and unwounded right half blade) were inoculated with different methods for each isolate, and the tests were repeated three times. Four days after inoculation, leaf spots were observed on all wounded leaves, while 5-10% of the unwounded leaves showed lesions. Control leaves inoculated with PDA discs and sterile water remained symptomless. Colletotrichum. siamense was re-isolated from the lesions, confirming Koch's postulates. At least 60 plant species have been reported to be infected by C. siamense worldwide (Ji et al. 2019). To our knowledge, this is the first report of C. siamense causing leaf spot on M. alba in China.

Arabidopsis PP6 phosphatases dephosphorylate PIF proteins to repress photomorphogenesis.

The PHYTOCHROME-INTERACTING FACTORs (PIFs) play a central role in repressing photomorphogenesis, and phosphorylation mediates the stability of PIF proteins. Although the kinases responsible for PIF phosphorylation have been extensively studied, the phosphatases that dephosphorylate PIFs remain largely unknown. Here, we report that seedlings with mutations in FyPP1 and FyPP3, 2 genes encoding the catalytic subunits of protein phosphatase 6 (PP6), exhibited short hypocotyls and opened cotyledons in the dark, which resembled the photomorphogenic development of dark-grown pifq mutants. The hypocotyls of dark-grown sextuple mutant fypp1 fypp3 (f1 f3) pifq were shorter than those of parental mutants f1 f3 and pifq, indicating that PP6 phosphatases and PIFs function synergistically to repress photomorphogenesis in the dark. We showed that FyPPs directly interacted with PIF3 and PIF4, and PIF3 and PIF4 proteins exhibited mobility shifts in f1 f3 mutants, consistent with their hyperphosphorylation. Moreover, PIF4 was more rapidly degraded in f1 f3 mutants than in wild type after light exposure. Whole-genome transcriptomic analyses indicated that PP6 and PIFs coregulated many genes, and PP6 proteins may positively regulate PIF transcriptional activity. These data suggest that PP6 phosphatases may repress photomorphogenesis by controlling the stability and transcriptional activity of PIF proteins via regulating PIF phosphorylation.

Density functional studies of the stepwise substitution of pyridine, pyridazine, pyrimidine, pyrazine, and 1,3,5-triazine with BCO.

The structures, stabilities, and aromaticities of a series of (BCO) n (CH)5-n N (n = 0-5), (BCO) n (CH)4-n N2 (n = 0-4), and 1,3,5-(BCO) n (CH)3-n N3 (n = 0-3) clusters were investigated at the B3LYP density functional level of theory. The most stable positional isomers of individual clusters were obtained. All of the calculated CO binding energies were positive, suggesting that the BCO-substituted species are stable. It was found that the BCO-substituted structures are much less strained than their carbocation counterparts. The negative nucleus-independent chemical shifts (NICSs) obtained show that all of the BCO-substituted species possess three-dimensional aromaticity, in good accord with the aromaticities of the corresponding hydrocarbon species.

Density functional studies of the stepwise substitution of pyrrole, furan, and thiophene with BCO.

The structures, stabilities, and aromaticities of a series of (BCO) n (CH)4-n NH (n = 0-4), (BCO) n (CH)4-n O (n = 0-4), and (BCO) n (CH)4-n S (n = 0-4) clusters were investigated at the B3LYP density functional level of theory. The most stable positional isomers of the individual clusters were obtained. All of the calculated CO binding energies were exothermic, suggesting that these BCO-substituted species are stable. Calculated differences in strain energy between the BCO-substituted structures and their corresponding hydrocarbon clusters were all exothermic, indicating that the BCO-substituted structures are less strained. The negative nucleus-independent chemical shift (NICS) values obtained show that these BCO-substituted clusters are aromatic compounds, in good agreement with the aromaticities of the corresponding hydrocarbon species. To aid further experimental investigations, CO-stretching frequencies were also computed.

Structures and aromaticity of Cationic closo-BnHn-3(CO)3+ (n = 5-12).

Structures and stabilities of tricarbonyl closo-boranes cation, BnHn-3(CO)3+ (n = 5-12), isolobal with cationic closo-carboranes C3Bn-3Hn+, have been investigated at the B3LYP/6-311+G** level of theory. The most stable positional isomers of individual cluster are in agreement with those of closo-C3Bn-3Hn+ clusters except for n = 8 and 10. Energetic analysis identifies closo-B6H3(CO)3+, closo-B10H7(CO)3+ and closo-B12H9(CO)3+ as the most stable cages. It is also found that closo-BnHn-3(CO)3+ is much less strained than closo-C3Bn-3Hn+. The negative nucleus independent chemical shifts (NICS) at the cage center reveal three-dimensional aromaticity of the closo-BnHn-3(CO)3+ cages. The CO stretching frequencies have been computed in advance to aid experimental study.

Boron nitride cages from B12N12 to B36N36: square-hexagon alternants vs boron nitride tubes.

The structures and stabilities of square-hexagon alternant boron nitrides (Bx Nx , x=12-36) vs their tube isomers containing octagons, decagons and dodecagons have been computed at the B3LYP density functional level of theory with the correlation-consistent cc-pVDZ basis set of Dunning. It is found that octagonal B20N20 and B24N24 tube structures are more stable than their square-hexagon alternants by 18.6 and 2.4 kcal mol(-1), respectively, while the square-hexagon alternants of other cages are more stable. Trends in stability as a function of cluster size are discussed.

Structures and energies of isolobal (BCO)n and (CH)n cages.

The structures and energies of isolobal (CH)n and (BCO)n polyhedral species, computed at the B3LYP density functional theory level, reveal contrasts in behavior. The strain energies of the (BCO)n cages are much smaller. Also unlike the (CH)n cages, the most stable (BCO)n polyhedra (n > or = 10) prefer structures with the largest number of three-membered rings. The planar (or nearly planar) faces of the cage systems were modeled by computations on planar, isoelectronic (CH2)n (Dnh) and (HBCO)n (Cnv) rings. While the strain energies of all the planar carbon rings, relative to the most stable D5h (CH2)5, were large, the strain energies of all the planar (HBCO)n (Cnv) rings were small. Remarkably, the three-membered (HBCO)3 (C3v) ring was the most stable. Finally, large (BCO)n systems prefer tubelike rather than cage structures.