FEBS Letters

doi: 10.1002/1873-3468.14124pmid: N/A

Bhunia, Rupam Kumar; Menard, Guillaume N.; Eastmond, Peter J.

doi: 10.1002/1873-3468.14365pmid: 35490366

Achieving gain‐of‐function phenotypes without inserting foreign DNA is an important challenge for plant biotechnologists. Here, we show that a gene can be brought under the control of a promoter from an upstream gene by deleting the intervening genomic sequence using dual‐guide CRISPR/Cas9. We fuse the promoter of a nonessential photosynthesis‐related gene to DIACYLGLYCEROL ACYLTRANSFERASE 2 (DGAT2) in the lipase‐deficient sugar‐dependent 1 mutant of Arabidopsis thaliana to drive ectopic oil accumulation in leaves. DGAT2 expression is enhanced more than 20‐fold and the triacylglycerol content increases by around 30‐fold. This deletion strategy offers a transgene‐free route to engineering traits that rely on transcriptional gain‐of‐function, such as producing high lipid forage to increase the productivity and sustainability of ruminant farming.

Phan, Kieu Anh Thi; Paeng, Seol Ki; Chae, Ho Byoung; Park, Joung Hun; Lee, Eun Seon; Wi, Seong Dong; Bae, Su Bin; Kim, Min Gab; Yun, Dae‐Jin; Kim, Woe‐Yeon; Lee, Sang Yeol

doi: 10.1002/1873-3468.14410pmid:

Marzoll, Daniela; Serrano, Fidel E.; Diernfellner, Axel C. R.; Brunner, Michael

doi: 10.1002/1873-3468.14435pmid: 35735764

Timing by the circadian clock of Neurospora is associated with hyperphosphorylation of frequency (FRQ), which depends on anchoring casein kinase 1a (CK1a) to FRQ. It is not known how CK1a is anchored so that approximately 100 sites in FRQ can be targeted. Here, we identified two regions in CK1a, p1 and p2, that are required for anchoring to FRQ. Mutation of p1 or p2 impairs progressive hyperphosphorylation of FRQ. A p1‐mutated strain is viable but its circadian clock is non‐functional, whereas a p2‐mutated strain is non‐viable. Our data suggest that p1 and potentially also p2 in CK1a provide an interface for interaction with FRQ. Anchoring via p1‐p2 leaves the active site of CK1a accessible for phosphorylation of FRQ at multiple sites.

Qin, Zheng; Wu, Ya‐Nan; Sun, Tian‐Tian; Ma, Ting; Xu, Meng; Pang, Chen; Li, Shan‐Wei; Li, Sha

doi: 10.1002/1873-3468.14422pmid: 35680649

The development of male and female gametophytes is a prerequisite for successful propagation of angiosperms. The small GTPases RAN play fundamental roles in numerous cellular processes. Although RAN GTPases have been characterized in plants, their roles in cellular processes are far from understood. We report here that RAN GTPases in Arabidopsis are critical for gametophytic development. RAN1 loss‐of‐function showed no defects in gametophytic development likely due to redundancy. However, the expression of a dominant negative or constitutively active RAN1 resulted in gametophytic lethality. Genetic interference of RAN GTPases caused the arrest of pollen mitosis I and of mitosis of functional megaspores, implying a key role of properly regulated RAN activity in mitosis during gametophytic development.

Zhang, Rui; Dong, Qiuyan; Zhao, Panpan; Eickelkamp, Anna; Ma, Chunmin; He, Gefeng; Li, Fangjun; Wallrad, Lukas; Becker, Tobias; Li, Zhaohu; Kudla, Jörg; Tian, Xiaoli

doi: 10.1002/1873-3468.14377pmid: 35561107

Efficient allocation of the essential nutrient potassium (K+) is a central determinant of plant ion homeostasis and involves AKT2 K+ channels. Here, we characterize four AKT2 K+ channels from cotton and report that xylem and phloem expressed GhAKT2bD facilitates K+ allocation and that AKT2‐silencing impairs plant growth and development. We uncover kinase activity‐dependent activation of GhAKT2bD‐mediated K+ uptake by AtCBL4–GhCIPK1 calcium signalling complexes in HEK293T cells. Moreover, AtCBL4–AtCIPK6 complexes known to convey activation of AtAKT2 in Arabidopsis also activate cotton GhAKT2bD in HEK293T cells. Collectively, these findings reveal an essential role for AKT2 in the source‐sink allocation of K+ in cotton and identify GhAKT2bD as subject to complex regulation by CBL–CIPK Ca2+ sensor–kinase complexes.

Furubayashi, Maiko; Maoka, Takashi; Mitani, Yasuo

doi: 10.1002/1873-3468.14342pmid: 35344590

Carotenoids with rare 6‐hydroxy‐3‐keto‐ε‐end groups, such as piprixanthin, vitixanthin, or cochloxanthin, found in manakin birds or plants, are rare carotenoids with high antioxidant activity. The same chemical structure is found in abscisic acid or blumenol, apocarotenoids found in plants or fungi. In this study, we serendipitously discovered that the promiscuous activity of the β‐carotene hydroxylase CrtZ, a diiron‐containing membrane protein, can catalyze the formation of 6‐hydroxy‐3‐keto‐ε‐end by using epoxycarotenoids antheraxanthin or violaxanthin as substrate. We suggest that the reaction mechanism is similar to that of a rhodoxanthin biosynthetic enzyme. Our results provide a further understanding of the reaction mechanism of diiron‐containing β‐carotene hydroxylases, as well as insight into the biosynthesis of natural compounds with 6‐hydroxy‐3‐keto‐ε‐end carotenoid derivatives.

Dilokpimol, Adiphol; Verkerk, Bart; Li, Xinxin; Bellemare, Annie; Lavallee, Mathieu; Frommhagen, Matthias; Underlin, Emilie Nørmølle; Kabel, Mirjam A.; Powlowski, Justin; Tsang, Adrian; Vries, Ronald P.

doi: 10.1002/1873-3468.14322pmid: 35187647

Feruloyl esterases (FAEs) and acetyl xylan esterases (AXEs) are important enzymes for plant biomass degradation and are both present in Carbohydrate Esterase family 1 (CE1) of the Carbohydrate‐Active enZymes database. In this study, ten novel fungal CE1 enzymes from different subfamilies were heterologously produced and screened for their activity towards model and complex plant biomass substrates. CE1_1 enzymes possess AXE activity, while CE1_5 enzymes showed FAE activity. Two enzymes from CE1_2 and one from CE1_5 possess dual feruloyl/acetyl xylan esterase (FXE) activity, showing expansion of substrate specificity. The new FXEs from CE1 can efficiently release both feruloyl and acetyl residues from feruloylated xylan, making them particularly interesting novel components of industrial enzyme cocktails for plant biomass degradation.

Matsuzawa, Tomohiko; Watanabe, Masahiro; Nakamichi, Yusuke; Akita, Hironaga; Yaoi, Katsuro

doi: 10.1002/1873-3468.14427pmid: 35717558

Aspergillus oryzae isoprimeverose‐producing oligoxyloglucan hydrolase (IpeA) releases isoprimeverose units (α‐d‐xylopyranosyl‐(1→6)‐d‐glucose) from the non‐reducing end of xyloglucan oligosaccharides and belongs to glycoside hydrolase family 3. In this paper, we report the X‐ray crystal structure of the IpeA complexed with a xyloglucan oligosaccharide, (XXXG: Glc4Xyl3). Trp515 of IpeA plays a critical role in XXXG recognition at positive subsites. In addition, docking simulation of IpeA‐XXXG suggested that two Tyr residues (Tyr268 and Tyr445) are involved in the catalytic reaction mechanism of IpeA. Tyr268 plays an important role in product turnover, whereas Tyr445 stabilizes the acid/base Glu524 residue, which serves as a proton donor. Our findings indicate that the substrate recognition machinery of IpeA is specifically adapted to xyloglucan oligosaccharides.

Kalapos, Miklós Péter; Bari, Lidia

doi: 10.1002/1873-3468.14408pmid: 35599367

Glycolysis is present in nearly all organisms alive today. This article proposes an evolutionary trajectory for the development of glycolysis in the framework of the chemoautotrophic theory for the origin of life. In the proposal, trioses and triose‐phosphates were appointed to starting points. The six‐carbon and the three‐carbon intermediates developed in the direction of gluconeogenesis and glycolysis, respectively, differing from the from‐bottom‐to‐up development of enzymatic glycolysis. The examination of enzymatic reaction mechanisms revealed that the enzymes incorporated chemical mechanisms of the nonenzymatic stage, making possible to identify kinship between glyoxalases and glyceraldehyde 3‐phosphate dehydrogenase as well as methylglyoxal synthase and triose‐phosphate isomerase. This developmental trajectory may shed light on how glycolysis might have developed in the nonenzymatic era.