A calcium-dependent protein kinase and an E3 ubiquitin ligase fine-tune rice immunity

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A calcium-dependent protein kinase and an E3 ubiquitin ligase fine-tune rice immunity

Mou et al. explore the growth­–defense trade-off in rice.

https://doi.org/10.1093/plcell/koad265

Baohui Mou, Jiyang Wang, and Wenxian Sun

China Agricultural University

Background: Plants need to keep a balance between growth and defense against diseases. How plants fine-tune this trade-off remains largely a mystery. We identified two protein components in rice disease resistance, a calcium-dependent protein kinase OsCPK17 and an E3 ubiquitin ligase OsPUB12. OsCPK17 promotes accumulation of another immune protein, OsRLCK176, and helps rice plants fight off various diseases. On the other hand, OsPUB12 breaks down OsRLCK176 to prevent excessive immunity. We wanted to understand how the two immune regulators, OsCPK17 and OsPUB12, work together to keep rice plants robust and healthy.

Question: We first aimed to answer the following question: How does the calcium-dependent protein kinase OsCPK17 regulate rice defenses against pathogen attacks? To answer this question, we identified two other proteins, OsRLCK176 and OsPUB12, through protein–protein interaction screening. Next, we asked: How do these proteins work together to control disease resistance in rice?

Findings: In rice, OsRLCK176 is a central player in disease resistance. To balance growth and defense in rice, OsRLCK176 is under tight control by different immune components. We revealed that a calcium-dependent protein kinase OsCPK17 functions as a “boost” button in the rice immune system. When a pathogen threat appears, OsCPK17 is activated to promote OsRLCK176 accumulation and thereby enhance basal defense in rice. However, without danger signals, the E3 ubiquitin ligase OsPUB12 targets OsRLCK176 for recycling, thus keeping basal defense low. We reveal that OsCPK17, OsPUB12 and OsRLCK176 function as a team to fine-tune trade-offs between rice growth and defense.

Next steps: The calcium-dependent protein kinases OsCPK17 and OsCPK4 function as positive and negative regulators through interacting with OsRLCK176, respectively. Future research should examine the molecular mechanisms by which the two calcium sensors function in seemingly opposite ways in rice resistance and aim to create disease-resistant rice plants based on the uncovered immune mechanisms.

Baohui Mou, Guosheng Zhao, Jiyang Wang, Shanzhi Wang, Feng He, Yuese Ning, Dayong Li, Xinhang Zheng, Fuhao Cui, Fang Xue, Shiyong Zhang, Wenxian Sun. (2023). The OsCPK17-OsPUB12-OsRLCK176 module regulates immune homeostasis in rice. https://doi.org/10.1093/plcell/koad265

Cytokinin signaling determines rice panicle size

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Chun, Fang, Savelieva, Lomin et al. explore the mechanism by which cytokinin signaling influences the size of rice panicle.

https://doi.org/10.1093/plcell/koad257

By Yan Chun (淳雁), and Xueyong Li (李学勇)

National Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, China

Background: The plant hormone cytokinin (CK) promotes plant meristem initiation and maintenance. A high level of CK in rice can enhance meristem activity and enlarge panicles, while a low level of CK can cause premature termination of the meristem and result in smaller panicles. The CK signal is transmitted from histidine kinase (HK) receptors to phosphotransfer proteins (HPts) and response regulators (RRs). The roles of several CK receptors and RRs in rice panicle development have been partially revealed, but their regulatory relationships and downstream factors remain unclear.

Question: Which RR receives the CK signal from the CK receptor OHK4? Which genes controlling panicle size are influenced by RRs? Are there any regulators that affect OHK4?

Findings: We discovered that a loss-of-function mutation of OHK4 disrupts CK signaling and reduces rice panicle size. The phosphorylation level of the type-B RR OsRR21 is dependent on OHK4. Overexpression of OsRR21 partially rescued the short panicle phenotype of the ohk4 mutant. IDEAL PLANT ARCHITECTURE1 (IPA1)/WEALTHY FARMER’S PANICLE (WFP), encoding a positive regulator of rice panicle size, is directly activated by OsRR21. Interestingly, OHK4 expression is upregulated in the gain-of-function mutant ipa1-D but downregulated in loss-of-function ipa1 mutants. Genetic and molecular analyses revealed that IPA1/WFP regulates OHK4 through interactions with two TCP transcription factors. Therefore, OHK4, OsRR21, and IPA1/WFP form a positive feedback loop to control meristem activity and panicle development in rice.

Next steps: Although the connection between OHK4 and OsRR21 has been revealed, we still do not know which histidine phosphotransfer proteins (HPts) act as intermediates in this process. The relationship between OHK4 and other RRs is also unclear. Whether OsRR21 regulates other meristem or panicle regulators is quite an intriguing question.

Yan Chun, Jingjing Fang, Ekaterina M. Savelieva, Sergey N. Lomin, Jiangyuan Shang, Yinglu Sun, Jinfeng Zhao, Ashmit Kumar, Shoujiang Yuan, Xue-Feng Yao, Chun-Ming Liu, Dmitry V. Arkhipov, Georgy A. Romanov, and Xueyong Li. (2023). The cytokinin receptor OHK4/OsHK4 regulates inflorescence architecture in rice via an IDEAL PLANT ARCHITECTURE1/WFP-mediated positive feedback circuit. https://doi.org/10.1093/plcell/koad257

Examining apical floral decline in the indeterminate inflorescence apex of barley

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Shanmugaraj et al. used spatiotemporal multi-omics studies to elucidate the mechanism of apical degeneration of barley inflorescence meristem and proposes a molecular framework behind this process, the manipulation of which may increase yield in barley and other related cereals.

Nandhakumar Shanmugaraj1, Thorsten Schnurbusch1,2

1Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstr. 3, OT Gatersleben, 06466 Seeland, Germany

2Martin Luther University Halle-Wittenberg, Faculty of Natural Sciences III, Institute of Agricultural and Nutritional Sciences, 06120 Halle, Germany

Background: In many cereal crops, some floret primordia develop into fertile florets that form grains, but some fail to form grains. In barley (Hordeum vulgare L.), arrest and cessation of the inflorescence apex is usually followed by death of apical spikelets/florets and the central axis beneath the apex during early reproductive development. Previous studies on apical floret death have proposed prominent roles for assimilate allocation, phytohormone homeostasis, and poor vascular connections; however, the underlying molecular mechanism of pre-anthesis tip degeneration (PTD) in barley remains elusive. Reducing the extent of spikelet/floret loss represents an opportunity to increase grain yield in barley and related cereals.

Question: Is PTD of the barley inflorescence developmentally programmed? Is the apical part of the barley inflorescence limited by primary metabolites? Does the dying apical part display distinct phytohormone patterning compared to the viable parts? What are the major gene players regulating PTD?

Findings: Our spatiotemporal multi-omics studies revealed that barley inflorescence PTD is accompanied by sugar depletion, amino acid degradation, and abscisic acid responses involving transcriptional regulators of senescence. Photosynthesis, inflorescence greening, and energy metabolism contribute to proper spikelet growth and differentiation and were restricted to viable parts. The transcription factor GRASSY TILLERS1 (HvGT1) was identified as a modulator of barley PTD: a HvGT1 knockout showed delayed PTD, harbored more differentiated apical spikelets and exhibited an increased final spikelet number. This study proposes a molecular framework for barley spike PTD, the manipulation of which may increase yield in barley and other related cereals.

Next steps: Loss of HvGT1 function delayed, but did not abolish, PTD. What are the upstream regulators defining the onset and extent of PTD? Putative PTD candidates can be used to exploit their natural allelic variants, their functional validation, and multiplex editing to increase grain yield in barley and other related cereals.

Reference: 

Nandhakumar Shanmugaraj, Jeyaraman Rajaraman, Sandip Kale, Roop Kamal, Yongyu Huang, Venkatasubbu Thirulogachandar, Adriana Garibay-Hernández, Nagaveni Budhagatapalli, Yudelsy Antonia Tandron Moya1, Mohammed R. Hajirezaei1, Twan Rutten1, Götz Hensel, Michael Melzer, Jochen Kumlehn, Nicolaus von Wirén, Hans-Peter Mock and Thorsten Schnurbusch (2023). Multilayered regulation of developmentally programmed pre-anthesis tip degeneration of the barley inflorescence. https://doi.org/10.1093/plcell/koad164

DRIF1 and SORTING NEXIN 1 regulate membrane protein homeostasis in Arabidopsis

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Zhu et al. explored the function of DRIF1 as a FREE1 suppressor in Arabidopsis. The Plant Cell (2023).

https://doi.org/10.1093/plcell/koad227

By Ying Zhu1, Jinbo Shen4 and Liwen Jiang1,2,3

1School of Life Sciences, Centre for Cell & Developmental Biology and State Key Laboratory of Agrobiotechnology, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China.

2Institute of Plant Molecular Biology and Agricultural Biotechnology, The Chinese University of Hong Kong, Shatin, Hong Kong, China.

3CUHK Shenzhen Research Institute, Shenzhen 518057, China.

4State Key Laboratory of Subtropical Silviculture, Zhejiang A&F University, Hangzhou 311300, China.

Background: Cells maintain the balance and stability of membrane proteins through vacuolar degradation and recycling, which is crucial for plant growth and interactions with the environment. Previously we characterized an endosomal sorting complex required for transport (ESCRT) protein called FYVE DOMAIN PROTEIN REQUIRED FOR ENDOSOMAL SORTING 1 (FREE1) in Arabidopsis thaliana. FREE1 performs multiple functions in plant cells, such as regulation of organelle biogenesis and vacuolar degradation of membrane proteins. Via a forward genetic screening approach using the FREE1-RNAi mutant line, we recently identified and characterized two suppressor of free1 (sof) mutants with causal genes called BRO1-DOMAIN PROTEIN AS FREE1 SUPPRESSOR (BRAF) and RESURRECTION1 (RST1).

Questions: What are the underlying mechanisms and regulators of FREE1-mediated functions in plants?

Findings: In this genetic screen, we further identified two sof mutants, sof10 and sof641. These mutants had mutations in a protein called DEAH AND RING DOMAIN-CONTAINING PROTEIN AS FREE1 SUPPRESSOR 1 (DRIF1). We showed that DRIF1 has a close homolog, DRIF2, in the Arabidopsis genome. The embryos of drif1 drif2 double mutants arrested at the globular stage and formed enlarged multivesicular bodies with increased numbers of intraluminal vesicles. DRIF1 coordinates with SORTING NEXIN 1 to regulate PIN-FORMED2 recycling to the plasma membrane. Overall, through a combination of cellular, biochemical, and genetic approaches, our study revealed DRIF1 functions in orchestrating FREE1-mediated intraluminal vesicle formation of multivesicular bodies and vacuolar sorting of membrane proteins for degradation in plants.Next steps: Future studies will aim to understand the mechanisms underlying the formation of enlarged multivesicular bodies with increased intraluminal vesicles in drif1 drif2 double mutants, and how DRIF regulate SORTING NEXIN 1-dependent membrane protein recycling to the plasma membrane in response to multiple environmental cues.

Reference:

Ying Zhu, Qiong Zhao, Wenhan Cao, Shuxian Huang, Changyang Ji, Wenxin Zhang, Marco Trujillo, Jinbo Shen, Liwen Jiang. (2023). The plant-unique DRIF1 regulates membrane protein homeostasis in coordination with SNX1. https://doi.org/10.1093/plcell/koad227

 

A prolyl 4-hydroxylase gene contributes to stem growth in poplar

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Xiao, Fang, Zhang, et al. identify a gene affecting stem growth in poplar.

Liang Xiao (Beijing University of Agriculture and Beijing Forestry University), Yuanyuan Fang (Beijing Forestry University), He Zhang (Peking University), and Deqiang Zhang (Beijing University of Agriculture and Beijing Forestry University)

https://doi.org/10.1093/plcell/koad212

Background: Forest trees are long-lived, woody perennials, making them dramatically different from Arabidopsis and other herbaceous annual model plants. Poplar (Populus) offers the opportunity to study biological questions pertinent to perennial stem growth. The annual cycle of perennial growth is synchronized with the seasons, based on periodic environmental fluctuations. Thus, perennial growth requires sophisticated regulatory mechanisms to initiate growth in the meristems and facilitate annual growth. While the genetic basis of stem growth has been elucidated, the dynamic genetic architecture of perennial stem growth has remained largely unknown.

Question: Which genes are involved in the regulation of stem growth during the long life span of trees?

Findings: Based on the diameter at breast height (DBH) measured over eight years, we modeled the growth trajectories from 303 natural accessions of Populus tomentosa and identified a causal gene PtoP4H9 responsible for natural variation of DBH. The allelic variants in PtoP4H9 promoter altered the expression of PtoP4H9 by affecting the binding affinities of the transcription factor PtoBPC1. Genetic analysis showed that PtoP4H9 increased the modification abundance of O-arabinosides and decreased the mechanical rigidity of cell wall to promote cell expansion and stem radial growth in Populus. More importantly, we found that PtoP4H9 underwent strong natural selection during local adaptation of Populus tomentosa.

Next steps: Additional efforts will concentrate on the candidate genes responsible for a particular growth stage such as juvenile to adult phase change and senescence phase. Clarifying candidate gene function will enable us to understand the inter-annual variability of perennial stem growth and help predict forest productivity.

Reference:

Liang Xiao, Yuanyuan Fang, He Zhang, Mingyang Quan, Jiaxuan Zhou, Peng Li, Dan Wang, Li Ji, Pär K Ingvarsson, Harry X. Wu, Yousry A. El-Kassaby, Qingzhang Du, and Deqiang Zhang (2023) Natural variation in the prolyl 4-hydroxylase gene PtoP4H9 contributes to perennial stem growth in Populus. https://doi.org/10.1093/plcell/koad212

Phosphorylase enzyme required for small starch granules in wheat

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Nitin Uttam Kamble shows that the enzyme α-glucan phosphorylase interacts with a carbohydrate-binding protein to initiate the formation of small B-type starch granules in wheat.

Nitin Uttam Kamble, Farrukh Makhamadnojov, Brendan Fahy, David Seung

John Innes Centre, Norwich Research Park, NR4 7UH, UK

https://doi.org/10.1093/plcell/koad217

Background: Starch is the main storage carbohydrate in plants and a vital source of calories in human diets. In the Triticeae tribe, which includes wheat, barley and rye, starch forms two distinct types of granules in grain: large discoid A-type granules and small spherical B-type granules. The A-type granules form early during grain development and the B-type granules form later.

Question: We asked whether A- and B-type granules are initiated via similar or distinct biochemical mechanisms. To better understand the mechanism of B-type granule initiation, we looked for interaction partners of B-GRANULE CONTENT1 (BGC1), a carbohydrate-binding protein that is important for B-type granule initiation.

Findings: We discovered that BGC1 interacts with the α-glucan phosphorylase, PHS1, in developing wheat endosperm. PHS1 can efficiently elongate short maltooligosaccharides in vitro, but decades of research have failed to find a clear role for this enzyme in plants. We produced phs1 knockout mutants in wheat and discovered they had fewer B-type granules compared to the wild type. By examining granule formation during grain development in the mutant, as well as double mutant combinations with bgc1, we discovered that loss of PHS1 only affects the formation of B-type granules, not A-type granules. Our findings reveal an indispensable role for PHS1 in wheat and demonstrate that A- and B-type granule initiations occur through distinct biochemical mechanisms.

Next steps: We are investigating whether the other BGC1 interaction partners identified in our study are involved in B-type granule initiation and studying how the actions of these proteins are coordinated. Since B-type granules affect the nutritional and functional properties of wheat, we are testing our phs1 mutants in various industrial applications.

Reference:

Nitin Uttam Kamble, Farrukh Makhamadnojov, Brendan Fahy, Carlo Martins, Gerhard Saalbach, David Seung (2023) Initiation of B-type starch granules in wheat endosperm requires the plastidial α-glucan phosphorylase PHS1. https://doi.org/10.1093/plcell/koad217

What happens when DNA breaks are repaired and when repair fails?

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Samach et al. use a pigment-based visual assay to explore the repair of double-stranded DNA breakd in plants.

https://doi.org/10.1093/plcell/koad209

Avraham Levy and Aviva Samach, Weizmann Institute of Science, Rehovot, Israel.

Background:  Plant breeding is the art of combining desirable traits from parental genomes.  This normally occurs during meiosis through crossover, the exchange of chromosomal segments between homologous chromosomes.  So far it has not been possible to target the precise breakpoints of crossovers at meiosis. Somatic crossover is rare, but we previously showed that it can be stimulated by a DNA double-strand break (DSB) and can be transmitted to the next generation. This was shown in tomatoes (Solanum lycopersicum) at specific fruit-color loci, but a more general system is needed to become a useful tool for precise breeding.

Question:  To better understand somatic DSB repair via homologous recombination, we developed an assay enabling visualization of somatic crossover via segregation of a transgenic purple color marker (the Betalain pigment), at a broad range of loci.  Through this process, heterozygous tissues become homozygous, forming wild-type or dark purple sectors in tomato leaves, flowers or fruits.

Findings:  We confirmed that somatic crossover can be detected visually at a CRISPR-mediated DSB site, opening the prospect for precise breeding in crops.  Moreover, we showed that loss of heterozygosity could be due to major chromosomal rearrangements triggered by defective repair of the DSB. Plants where this occurred contained large deletions and translocations and were sterile, showing micronuclei as well as bridges in dicentric chromosomes, at meiosis, as described in McClintock’s Breakage-Fusion-Bridge Cycle triggered by transposons.  This genomic reshuffling is similar to chromothripsis in mammalian cells. Both crossover and chromothripsis events were rare but could be detected thanks to the visual assay.

Next steps:  It will be important to better understand what determines the fate of a DSB; when repair leads to crossover or alternatively, to chromothripsis. The new assay will also enable to study how targeted somatic crossover can become more efficient for breeding applications.

Reference:

 Aviva Samach, Fabrizio Mafessoni, Or Gross, Cathy Melamed-Bessudo, Shdema Filler-Hayut, Tal Dahan-Meir, Ziva Amsellem, Wojciech P. Pawlowski, and Avraham A. Levy (2023) CRISPR/Cas9-induced DNA breaks trigger crossover, chromosomal loss, and chromothripsis-like rearrangements. https://doi.org/10.1093/plcell/koad209

A liverwort view of gibberellin biosynthesis

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Sun et al. investigated genes related to gibberellin biosynthesis in the liverwort Marchantia polymorpha and found them to be critical for far-red light responses. The Plant Cell (2023). 

https://doi.org/10.1093/plcell/koad216

By Rui Sun and Takayuki Kohchi (Kyoto University)

Background: The emergence and diversification of plant hormones over the past 450 million years is a fascinating part of land plant evolution. Gibberellins are a group of plant hormones that promote growth and regulate many developmental processes in flowering plants, and the gibberellin biosynthesis pathway is conserved in vascular plants including ferns and lycophytes. The sister lineage of vascular plants, bryophytes, lacks the ability to produce canonical gibberellins but encodes early-step enzymes for producing gibberellin precursors. It is possible that genes for these enzymes are inherited from the common ancestor of all land plants, but their physiological roles are less clear in bryophytes, especially in the liverwort and hornwort lineages.

Question: Are gibberellin precursors used to synthesize plant hormones in liverworts? What is the physiological activity of this putative hormone?

Findings: By chemical analysis, we detected a gibberellin (GA) biosynthesis intermediate, GA12, in the liverwort Marchantia polymorpha, but no canonical bioactive gibberellins. Genetic mutants and enzymatic assays nailed down the major enzymes for the production of GA12 and its precursor, ent-kaurenoic acid. We hypothesize that both substrates are involved in the biosynthesis of an unknown gibberellin-related hormone, GAMp. Far-red enriched light, which indicates the presence of competitive neighbors in nature, promotes GA12 accumulation in M. polymorpha. The biosynthesis enzymes are also indispensable for the liverwort to carry out proper developmental responses, including changes in growth direction and acceleration of sexual reproduction. Interestingly, overall plant growth is enhanced in GAMp-deficient mutants, which is opposite to gibberellin deficiency phenotypes in flowering plants.

Next steps: We hope to find the exact chemical structure and the complete biosynthetic route of GAMp. Since the canonical gibberellin receptor is not conserved in bryophytes, we are also curious about how GAMp is perceived.

Reference: 

Rui Sun, Maiko Okabe, Sho Miyazaki, Toshiaki Ishida, Kiyoshi Mashiguchi, Keisuke Inoue, Yoshihiro Yoshitake, Shohei Yamaoka, Ryuichi Nishihama, Hiroshi Kawaide, Masatoshi Nakajima, Shinjiro Yamaguchi, Takayuki Kohchi. (2023). Biosynthesis of gibberellin-related compounds modulates far-red light responses in the liverwort Marchantia polymorpha. https://doi.org/10.1093/plcell/koad216

苔类植物为赤霉素生物合成提供新视野

最近，来自日本京都大学的孙芮等人在The Plant Cell在线发表了题为Biosynthesis of gibberellin-related compounds modulates far-red light responses in the liverwort Marchantia polymorpha的研究论文，探索了苔类植物地钱中的赤霉素合成相关基因及其生理功能。

01 背景回顾：

在陆地植物长达4.5亿年的演化历史中，植物激素的起源和多样性分化是不可或缺的一环。在被子植物中，赤霉素是一类重要的植物激素，调节多种生长发育过程。此前研究认为，赤霉素的完整合成通路在包括蕨类和小叶类植物在内的维管植物中是保守的；而维管植物的姐妹类群——苔藓植物中缺乏关键合成酶，无法通过经典途径产生有生理活性的赤霉素。然而，赤霉素合成途径中催化前期步骤的多种合成酶在苔藓植物的基因组里普遍存在，意味着它们可能继承自陆地植物的共同祖先。在苔藓植物特别是苔类和角苔类植物中，对这些酶的生理作用研究还相当有限。

02 科学问题

苔类植物是否利用部分赤霉素合成通路来产生植物激素？该通路可能的生理功能是什么？

03 研究发现

通过质谱分析，我们检测了苔类植物地钱（Marchantia polymorpha）中的赤霉素相关化合物，除合成中间体GA12外未检出其他常见赤霉素。利用遗传和生化分析，我们确定了地钱中GA12及其前体对映-贝壳杉烯酸（KA）的主要合成酶（即MpCPS、MpKS、MpKOL1和MpKAOL1），并提出KA和GA12可能参与合成一种未知的类赤霉素激素GAMp。在自然环境下，周围植物的竞争会造成光照中远红光组分的增加。实验条件下远红光富集会促使地钱提高GA12的积累，而缺乏赤霉素相关合成酶的地钱则无法完成对远红光的发育响应，既无法改变生长极性也无法快速进入有性生殖。有趣的是，在地钱中阻断GAMp的合成常常促进植物生长，跟被子植物中赤霉素缺乏造成的矮小表型截然相反。

04 展望未来

我们将继续探索GAMp的活性形式和完整合成途径。鉴于苔藓植物同样缺失被子植物中的经典赤霉素受体GID1，我们也希望了解GAMp的信号感受机制。

原文作者：Rui Sun et al.

翻译：Rui Sun, Haonan Bao (Kyoto University)

Special issue: Human-machine collaboration in plant biology

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This is an excellent article to wrap up this year and lead us into the future. Introducing a special issue of Plant Cell Physiology, Nakajima et al. summarize an exciting collection of papers that look at diverse ways that plant biology can be enhanced through “human-machine collaborations”. Some of these, like microscopes (machines that enhance human vision) are already widely used, whereas others have a futuristic aura, such as P-MIRU (Polarization Multispectral Imaging for Reflection from Biological Surfaces). Many of the approaches use trainable algorithms such as artificial intelligence (AI), explainable AI, and deep-learning to provide insights into plant development and physiology that go way beyond human capabilities. Examples include methods that track and trace cells in 4D (at the root tip, cotyledons, and zygote). Another useful machine is a portable tool for quantifying stomatal movements in intact leaves, greatly increasing the rate at which data can be collected. Several articles describe how machines can be trained to interpret huge sets of data and images, leading to observations beyond human capabilities. This is an exciting article to share with your students. (Summary by Mary Williams @PlantTeaching) Plant Cell Physiol. 10.1093/pcp/pcad144