Stanford research reveals mechanisms for plant cell stability in drought

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Water deficit resistance in plants has long been a topic of interest for cultivating reliable crops. Some plants can alter their above-ground structure to lock in moisture, while others develop deep, industrious roots that find hard-to-reach water sources. While such responses are obvious to the naked eye, we know little about how responses to environmental stress occur at the microscopic, cellular level.

Arabidopsis thaliana grown in the Dinneny lab. Image credit: Yue Rui

Over a hundred years ago, a German botanist named Karl Hecht documented plant cell membranes peeling away from the cell wall when deprived of water. Yet parts of the membrane remained attached to the wall and created a strange web of anchor points, which became known as “Hechtian structures.” The material and purpose of these structures mystified scientists until recently.

Now, a Stanford-led team has shown that these anchor points keep the membrane connected to the wall during water loss and that plant cells with more of them recover better once water returns.

The study, published today in Cell, describes how the “molecular machine” that builds the cell wall also forges these critical anchor points in the membrane. Achieving this in-depth look at the structures required lead author and postdoctoral scholar Yue Rui to examine the cells of plant roots through live-cell imaging, protein mapping, and comparison of genetic mutations.

“I find it very satisfying to take a process that has been characterized now for over 100 years and establish what the molecular basis of it is,” said José Dinneny, professor of biology in the School of Humanities and Sciences and senior author of the study. “The images are beautiful and the ability of Yue to resolve very fine-scale changes in cellular structure has been a joy and a gift to watch.”

Conducting ‘mutant surveys’
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Rui began exploring the purpose of Hechtian structures by comparing responses to water stress across wild type and genetically mutated varieties of Arabidopsis – a small, weedy plant with many similarities to common food and bioenergy crops. Like cells in our own bodies, plant cells are defined by a plasma membrane that holds the internal components that perform important cellular functions. In addition, plant cells are encased in a cell wall, like “a balloon in a box,” according to Dinneny. Normally, the balloon is inflated with water and solutes that press against the wall. When the plant cell is exposed to stress and loses water, it’s like the pressure is released from the balloon. However, in plant cells, the membrane does not fully separate and parts of the balloon remain tethered to the walls of the box.

From left to right: Plant cell with water. Plant cell under stress. Plant cell under stress without wall-membrane tethers. Image credit: Yue Rui et al.

A plant cell can be modeled as a balloon in a box, where the plasma membrane and inner cell content are the balloon and its contents and the cell wall is the glass box. Under non-stress conditions the balloon is oppressed to the cell wall and held in place by cell wall-membrane connections. Under water deficit stress, water leaves the cell, here modeled as a balloon that has been partially deflated, but remaining partially inflated due to the presence of the attachment sites. However, when these attachment sites are removed, the cell loses more water, here modeled as a balloon that deflates more completely. | José Dinneny using ChatGPT

To look more closely at these sticky tethers, Rui partnered with Peter Dahlberg, assistant professor in the Photon Science Directorate at SLAC National Accelerator Laboratory and Stanford’s Department of Structural Biology in the School of Medicine, to conduct cryogenic electron tomography (cryoET) scans, an imaging technique that allows for 3D reconstructions of samples at near-atomic levels of detail.

“The cryoET imaging in this paper reflects the most advanced ways of exploring cell biology at the nanometer scale,” said Dinneny. “So this paper nicely bookends the utilization of advanced microscopy in biology from the initial observations of Karl Hecht to the observations of Hechtian structures using CryoET.”

Weaving resilience
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Rui observed that plants able to maintain greater numbers of tethers during stress recovered much better than those with fewer strands.

Clues to the molecular identity of the tethers came from characterizing differences between genetic strains. Plants with a cellulose deficiency mutation showed minimal root growth and the least resilience to stress, leading Rui and Dinneny to believe cellulose and the enzymes producing it were a key component in these anchoring threads. Live cell imaging, genetics, and protein mapping revealed the roles of two key proteins: the cellulose synthase complex (CSC) and remorins (REMs). These proteins work in opposite ways, with CSC strengthening the membrane’s attachment to the cell wall, while REMs act as a brake, limiting how many CSC proteins are present at each attachment site.

CSCs act like nanoscale weavers, stitching a thread of cellulose around the cell like a cocoon. As CSC lays down the cellulose threads, it also tethers the membrane to the wall during the stitching process. REMs, on the other hand, act as a hand that pulls them out, controlling how many stitches hold at any given time. When REM is missing, the number of CSCs in the membrane increases and more firmly anchors the membrane to the wall during stress. Identifying each protein’s role in this survival strategy opens up possibilities in bioengineering better crops. Cellular water loss is present in drought, salinity, heat, and freezing conditions, so understanding how plant cells cope with such water loss is more urgent than ever as climate swings grow more severe.

The future
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“For me, the next interesting direction is to observe this mechanism in species that are even more tolerant to drought and see if they have more stable or more dense membrane attachment sites,” said Rui. Future studies may also include examining these attachments in Arabidopsis across different stages of its life cycle, such as in dried seeds that can sit on a shelf for years yet still grow into plants later.

Overall, Dinneny is fascinated to find that these plant cells use cellulose both as a building material and as a lifeline.

Creative Nature
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“There’s a tinkering nature to life and to how organisms evolve,” said Dinneny. “Plant cells using the same protein machinery to build their cell walls but also to maintain cellular resilience under water deficit stress points to the multi-faceted creativity that is abundant in nature.”

Citation
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The study Plant cell wall-plasma membrane attachments mediate stress resilience through cellulose synthase complexes and remorins was published in Cell journal. Authors: Yue Rui, Magda Zaoralová, William P. Dwyer, Andrés V. Reyes, Tarabryn S. Grismer, Nikolaj B. Abel, Dharanidaran Jayachandran, Shishir P.S. Chundawat, Thomas Ott, Joseph J. Kieber, Peter D. Dahlberg, Shou-Ling Xu, José R. Dinneny

Additional co-authors are from Carnegie Institution for Science, Aarhus University, Rutgers University, the University of Freiburg, and the University of North Carolina. Dahlberg is also a principal investigator at the Stanford PULSE Institute and a member of Stanford Bio-X and the Wu Tsai Neurosciences Institute. Dinneny is also an investigator at the Howard Hughes Medical Institute and a member of Stanford Bio-X.

Funding
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The research was funded by the Life Sciences Research Foundation, the Department of Energy, the National Institutes of Health, the National Institute of General Medicine Sciences, the Stanford Precourt Institute for Energy, the Simons Foundation, the Howard Hughes Medical Institute, the German Research Foundation, the National Science Foundation, and the Carnegie endowment fund to the Carnegie mass spectrometry facility.