Disruption of three polyamine uptake transporter genes in rice by CRISPR/Cas9 gene editing confers tolerance to herbicide paraquat.

Weeds are a major biotic constraint that can cause dramatic crop production losses. Herbicide technology has been widely used by farmers as the most cost-effective weed control measure, and development of new strategy to improve herbicide tolerance in plants is urgently needed. The CRISPR/Cas9-based genome editing tool has been used in diverse applications related to agricultural technology for crop improvement. Here we identified three polyamine uptake transporter (PUT) genes in rice that are homologous to the Arabidopsis AtRMV1. We successfully demonstrate that CRISPR/Cas9-targeted mutagenesis of OsPUT1/2/3 greatly improves paraquat resistance in rice without obvious yield penalty. Therefore, manipulation of these loci could be valuable for producing transgene-free rice with improved herbicide resistance in future. Supplementary Information: The online version contains supplementary material available at 10.1007/s42994-022-00075-4.

Quantitative Proteomic Analysis of ER Stress Response Reveals both Common and Specific Features in Two Contrasting Ecotypes of Arabidopsis thaliana.

Accumulation of unfolded and misfolded proteins in endoplasmic reticulum (ER) elicits a well-conserved response called the unfolded protein response (UPR), which triggers the upregulation of downstream genes involved in protein folding, vesicle trafficking, and ER-associated degradation (ERAD). Although dynamic transcriptomic responses and the underlying major transcriptional regulators in ER stress response in Arabidopsis have been well established, the proteome changes induced by ER stress have not been reported in Arabidopsis. In the current study, we found that the Arabidopsis Landsberg erecta (Ler) ecotype was more sensitive to ER stress than the Columbia (Col) ecotype. Quantitative mass spectrometry analysis with Tandem Mass Tag (TMT) isobaric labeling showed that, in total, 7439 and 7035 proteins were identified from Col and Ler seedlings, with 88 and 113 differentially regulated (FC > 1.3 or <0.7, p < 0.05) proteins by ER stress in Col and Ler, respectively. Among them, 40 proteins were commonly upregulated in Col and Ler, among which 10 were not upregulated in bzip28 bzip60 double mutant (Col background) plants. Of the 19 specifically upregulated proteins in Col, as compared with that in Ler, components in ERAD, N-glycosylation, vesicle trafficking, and molecular chaperones were represented. Quantitative RT-PCR showed that transcripts of eight out of 19 proteins were not upregulated (FC > 1.3 or <0.7, p < 0.05) by ER stress in Col ecotype, while transcripts of 11 out of 19 proteins were upregulated by ER stress in both ecotypes with no obvious differences in fold change between Col and Ler. Our results experimentally demonstrated the robust ER stress response at the proteome level in plants and revealed differentially regulated proteins that may contribute to the differed ER stress sensitivity between Col and Ler ecotypes in Arabidopsis.

A membrane-associated NAC transcription factor OsNTL3 is involved in thermotolerance in rice.

Heat stress induces misfolded protein accumulation in endoplasmic reticulum (ER), which initiates the unfolded protein response (UPR) in plants. Previous work has demonstrated the important role of a rice ER membrane-associated transcription factor OsbZIP74 (also known as OsbZIP50) in UPR. However, how OsbZIP74 and other membrane-associated transcription factors are involved in heat stress tolerance in rice is not reported. In the current study, we discovered that OsNTL3 is required for heat stress tolerance in rice. OsNTL3 is constitutively expressed and up-regulated by heat and ER stresses. OsNTL3 encodes a NAC transcription factor with a predicted C-terminal transmembrane domain. GFP-OsNTL3 relocates from plasma membrane to nucleus in response to heat stress and ER stress inducers. Loss-of-function mutation of OsNTL3 confers heat sensitivity while inducible expression of the truncated form of OsNTL3 without the transmembrane domain increases heat tolerance in rice seedlings. RNA-Seq analysis revealed that OsNTL3 regulates the expression of genes involved in ER protein folding and other processes. Interestingly, OsNTL3 directly binds to OsbZIP74 promoter and regulates its expression in response to heat stress. In turn, up-regulation of OsNTL3 by heat stress is dependent on OsbZIP74. Thus, our work reveals the important role of OsNTL3 in thermotolerance, and a regulatory circuit mediated by OsbZIP74 and OsNTL3 in communications among ER, plasma membrane and nucleus under heat stress conditions.

Nectary tracks as pollinator manipulators: The pollination ecology of Swertia bimaculata (Gentianaceae).

Floral nectaries are closely associated with biotic pollination, and the nectar produced by corolla nectaries is generally enclosed in floral structures. Although some Swertia spp. (Gentianaceae), including S. bimaculata, evolved a peculiar form of corolla nectaries (known as "gland patches") arranged in a conspicuous ring on the rotate corolla and that completely expose their nectar, little is known about the pollination of these plants. Two hypotheses were made concerning the possible effects of gland patches: visual attraction and visitor manipulation. The floral traits, mating system, and insect pollination of S. bimaculata were examined, and the pollination effects of gland patches were evaluated. A comparative study was made using Swertia kouitchensis, a species with fimbriate nectaries. Swertia bimaculata flowers were protandrous, with obvious stamen movement leading to herkogamy in the female phase and to a significant reduction in nectary-anther distance. The species is strongly entomophilous and facultatively xenogamous. The daily reward provided per flower decreased significantly after the male phase. The most effective pollinators were large dipterans, and the visiting proportion of Diptera was significantly higher in S. bimaculata than in S. kouitchensis. Most visitors performed "circling behavior" in S. bimaculata flowers. Removing or blocking the nectaries caused no reduction in visiting frequency but a significant reduction in visit duration, interrupting the circling behavior. The circling behavior was encouraged by nectar abundance and promoted pollen dispersal. Visitor species with small body size had little chance to contact the anthers or stigma, revealing a filtration effect exerted by the floral design. These results rejected the "visual attraction" hypothesis and supported the "visitor manipulation" hypothesis. The nectary whorl within a flower acted like a ring-shaped track that urged nectar foragers to circle on the corolla, making pollination in S. bimaculata flowers more orderly and selective than that in classically generalist flowers.