The cytokinins have been implicated in many facets of plant growth and development including cell division and differentiation, shoot and root growth, apical dominance, senescence, fruit and seed development, and the response to biotic and abiotic stressors. Cytokinin levels are regulated by a balance between biosynthesis [isopentenyl transferase (IPT)], activation [Lonely Guy (LOG)], inactivation (O-glucosyl transferase), re-activation (β-glucosidase), and degradation [cytokinin oxidase/dehydrogenase (CKX)]. During senescence, the levels of active cytokinins decrease, with premature senescence leading to a decrease in yield. During the early stages of fruit and seed development, cytokinin levels are transiently elevated, and coincide with nuclear and cell divisions which are a determinant of final seed size. Exogenous application of cytokinin, ectopic expression of IPT, or down-regulation of CKX have, on occasions, led to increased seed yield, leading to the suggestion that cytokinin may be limiting yield. However, manipulation of cytokinins is complex, not only because of their pleiotropic nature but also because the genes coding for biosynthesis and metabolism belong to multigene families, the members of which are themselves spatially and temporally differentiated. Previous research on yield of rice showed that plant breeders could directly target the cytokinins. Modern genome editing tools could be employed to target and manipulate cytokinin levels to increase seed yield with the concurrent aim of maintaining quality. However, how the cytokinin level is modified and whether IPT or CKX is targeted may depend on whether the plant is considered to be in a source-limiting environment or to be sink limited.

J.S.)) and (Tel +64 275553582; email paula.jameson@canterbury.ac.nz (P.E.J.)) SummaryThe plant hormone group, the cytokinins, is implicated in both qualitative and quantitative components of yield. Cytokinins have opposing actions in shoot and root growth-actions shown to involve cytokinin dehydrogenase (CKX), the enzyme that inactivates cytokinin. We revise and provide unambiguous names for the CKX gene family members in wheat, based on the most recently released wheat genome database, IWGSC RefSeq v1.0 & v2.0. We review expression data of CKX gene family members in wheat, revealing tissue-specific gene family member expression as well as sub-genome-specific expression. Manipulation of CKX in cereals shows clear impacts on yield, root growth and orientation, and Zn nutrition, but this also emphasizes the necessity to unlink promotive effects on grain yield from negative effects of cytokinin on root growth and uptake of mineral nutrients, particularly Zn and Fe. Wheat is the most widely grown cereal crop globally, yet is under-research compared with rice and maize. We highlight gaps in our knowledge of the involvement of CKX for wheat. We also highlight the necessity for accurate analysis of endogenous cytokinins, acknowledging why this is challenging, and provide examples where inadequate analyses of endogenous cytokinins have led to unjustified conclusions. We acknowledge that the allohexaploid nature of bread wheat poses challenges in terms of uncovering useful mutations. However, we predict TILLING followed by whole-exome sequencing will uncover informative mutations and we indicate the potential for stacking mutations within the three genomes to modify yield components. We model a wheat ideotype based on CKX manipulation. 614

BackgroundAs the global population continues to expand, increasing yield in bread wheat is of critical importance as 20% of the world’s food supply is sourced from this cereal. Several recent studies of the molecular basis of grain yield indicate that the cytokinins are a key factor in determining grain yield. In this study, cytokinin gene family members in bread wheat were isolated from four multigene families which regulate cytokinin synthesis and metabolism, the isopentenyl transferases (IPT), cytokinin oxidases (CKX), zeatin O-glucosyltransferases (ZOG), and β-glucosidases (GLU). As bread wheat is hexaploid, each gene family is also likely to be represented on the A, B and D genomes. By using a novel strategy of qRT-PCR with locus-specific primers shared among the three homoeologues of each family member, detailed expression profiles are provided of family members of these multigene families expressed during leaf, spike and seed development.ResultsThe expression patterns of individual members of the IPT, CKX, ZOG, and GLU multigene families in wheat are shown to be tissue- and developmentally-specific. For instance, TaIPT2 and TaCKX1 were the most highly expressed family members during early seed development, with relative expression levels of up to 90- and 900-fold higher, respectively, than those in the lowest expressed samples. The expression of two cis-ZOG genes was sharply increased in older leaves, while an extremely high mRNA level of TaGLU1-1 was detected in young leaves.ConclusionsKey genes with tissue- and developmentally-specific expression have been identified which would be prime targets for genetic manipulation towards yield improvement in bread wheat breeding programmes, utilising TILLING and MAS strategies.

Background and Aims Pisum sativum L. (pea) seed is a source of carbohydrate and protein for the developing plant. By studying pea seeds inoculated by the cytokinin-producing bacterium, Rhodococcus fascians, we sought to determine the impact of both an epiphytic (avirulent) strain and a pathogenic strain on source-sink activity within the cotyledons during and following germination.Methods Bacterial spread was monitored microscopically, and real-time reverse transcription-quantitative PCR was used to determine the expression of cytokinin biosynthesis, degradation and response regulator gene family members, along with expression of family members of SWEET, SUT, CWINV and AAP genes -gene families identified initially in pea by transcriptomic analysis. The endogenous cytokinin content was also determined.Key Results The cotyledons infected by the virulent strain remained intact and turned green, while multiple shoots were formed and root growth was reduced. The epiphytic strain had no such marked impact. Isopentenyl adenine was elevated in the cotyledons infected by the virulent strain. Strong expression of RfIPT, RfLOG and RfCKX was detected in the cotyledons infected by the virulent strain throughout the experiment, with elevated expression also observed for PsSWEET, PsSUT and PsINV gene family members. The epiphytic strain had some impact on the expression of these genes, especially at the later stages of reserve mobilization from the cotyledons.Conclusions The pathogenic strain retained the cotyledons as a sink tissue for the pathogen rather than the cotyledon converting completely to a source tissue for the germinating plant. We suggest that the interaction of cytokinins, CWINVs and SWEETs may lead to the loss of apical dominance and the appearance of multiple shoots.

HighlightIncreases in seed yield must occur without impacting seed or plant quality. Specific CKX gene family members were identified as targets for breeders along with transporter and metabolic genes as quality markers.

The cytokinins, which are N 6 -substituted adenine derivatives, control key aspects of crop productivity. Cytokinin levels are controlled via biosynthesis by isopentenyl transferase (IPT), destruction by cytokinin oxidase/dehydrogenase (CKX), and inactivation via glucosylation by cytokinin glucosyl transferases (CGTs). While both yield components and tolerance to drought and related abiotic stressors have been positively addressed via manipulation of IPT and/or CKX expression, much less attention has been paid to the CGTs. As naming of the CGTs has been unclear, we suggest COGT, CNGT, CONGT and CNOGT to describe the O-, N-and dual function CGTs. As specific CGT mutants of both rice and arabidopsis showed impacts on yield components, we interrogated the wheat genome database, IWGSC RefSeq v1.0 & v2.0, to investigate wheat CGTs. Besides providing unambiguous names for the 53 wheat CGTs, we show their expression patterns in 70 developmental tissues and their response characteristics to various stress conditions by reviewing more than 1000 RNA-seq data sets. These revealed various patterns of responses and showed expression generally being more limited in reproductive tissues than in vegetative tissues. Multiple cis-regulatory elements are present in the 3 kb upstream of the start codons of the 53 CGTs. Elements associated with abscisic acid, light and methyl jasmonate are particularly over-represented, indicative of the responsiveness of CGTs to the environment. These data sets indicate that CGTs have potential value for wheat improvement and that these could be targeted in TILLING or gene editing wheat breeding programmes.genes controlling the formation of the deactivated cytokinin Oand N-glucosides. After introducing the cytokinins more generally, this article focuses on the cytokinin O-and N-glucosyl transferases and their potential roles in modifying yield in bread wheat, under ambient and stress conditions. Cytokinin biosynthesis and metabolismNaturally occurring cytokinins, which are adenine derivatives, fall into two groups: those with an isoprenoid side chain and those with an aromatic one. The isoprenoid cytokinins are considered to be the predominant forms (Jaworek et al., 2019;Sakakibara, 2006). The aromatics are not covered in this review as, while not exhaustively looked for in all cereals, they were not detected in either maize leaves (Lacuesta et al., 2018) or in rice (Kudo et al., 2012), although low levels of 6-benzylamino purine were recently reported in wheat spikes (Jabło nski et al., 2020) and low levels of various aromatic metabolites have been detected in germinating seeds of maize and oats (Stirk et al., 2012).

Sophora is a woody genus of the Leguminosae in which an unusual order and process of floral organ development is often observed. The SEM results for Sophora tetraptera revealed precocious initiation of the carpel, delayed development of petals, and floral organ development interrupted by an unusual prolonged summer-autumn dormant period which occurred between organ initiation and organ differentiation. These observations provided an opportunity to track key floral identity genes over an extended developmental period. Homologues of LEAFY, APETALA1, PISTILLATA, and AGAMOUS were isolated from S. tetraptera. Real-time PCR enabled a simultaneous and quantitative analysis of both the temporal and spatial expression patterns of these four genes. Expression differences in the range of three to five orders of magnitude were detected between different genes and between different stages of flower development for the same gene. Although not functionally tested, the spatial expression patterns of the genes were consistent with expectations based on the ABC model of floral development. Their temporal expression patterns were consistent with the timing of flower initiation and the unusual order of organ development. Quantitatively, while the expression levels of the LFY homologue and the A-class gene were high during the periods of organ initiation and organ differentiation and low during the summer-autumn dormant period, high expression levels of the B- and C-class genes were detected only during the rapid, albeit delayed, phase of organ differentiation. Additionally, the sustained expression of the floral organ identity genes after differentiation reflects on-going roles for these genes during subsequent organ development.

Transporter genes and cytokinins are key targets for crop improvement. These genes are active during the development of the seed and its establishment as a strong sink. However, during germination, the seed transitions to being a source for the developing root and shoot. To determine if the sucrose transporter (SUT), amino acid permease (AAP), Sugar Will Eventually be Exported Transporter (SWEET), cell wall invertase (CWINV), cytokinin biosynthesis (IPT), activation (LOG) and degradation (CKX) gene family members are involved in both the sink and source activities of seeds, we used RT-qPCR to determine the expression of multiple gene family members, and LC-MS/MS to ascertain endogenous cytokinin levels in germinating Pisum sativum L. We show that genes that are actively expressed when the seed is a strong sink during its development, are also expressed when the seed is in the reverse role of being an active source during germination and early seedling growth. Cytokinins were detected in the imbibing seeds and were actively biosynthesised during germination. We conclude that, when the above gene family members are targeted for seed yield improvement, a downstream effect on subsequent seed germination or seedling vigour must be taken into consideration.